Effects of elevated nutrients on heterotrophic protists and phytyoplankton in and around costal waters

In addition to what was covered in Tuesdays’ lecture, I looked further into the effects that nutrients entering the marine environment from land have on microbial communities, with regards to distributions of heterotrophic protists, and the impact of protist grazing on phytoplankton.

Heterotrophic nanoflagellates, ciliates, and heterotrophic dinoflagellates are important secondary producers in marine ecosystems. Studies have shown that on average, heterotrophic protists consume over 60% of global daily phytoplankton production.
One of the studies that I have looked at was the first to investigate heterotrophic protist community distribution and grazing impacts in the northern East China Sea (ECS), which has dynamic hydrographic conditions. The shallower western shelf water has lower salinity, higher nutrient concentrations, and higher phytoplankton stocks. These conditions are driven by the East China Sea Current (ECSC), which incorporates water diluted by Chanjiang river water (CDW). The deeper eastern section of the ECS has lower nutrient concentrations due to the year-round persistence of the Tsushima Warm Current (TWC).
The planktonic food web structure in this region can be significantly affected by the CDW, which drives distribution of biological and chemical properties in this body of water. Changes in season affect the amount of CDW present in the ESC, with inflow of river water being primarily determined by seasonal wind direction.

Five cruises were conducted over four seasons, on which environmental and biological properties were measured including the abundance and composition of heterotrophic protists, particulate organic carbon, particulate organic nitrogen, suspended solids, and chlorophyll a. Measurements and samples were taken over a range of depths and a number of locations from the water body. water temperature and salinity measurements were also taken. A dilution method measuring the total chlorophyll a concentration was used to measure specific phytoplankton growth rates and protist grazing.

Patchy distributions of biomass were observed on all cruises, with a generally higher biomass being observed in waters with higher nutrient levels and lower salinity in western areas affected by the Changjiang river discharge. These results match similar patterns where heterotophic protists are documented to have an inverse relationship with salinity. Numerically, small heterotrophic dinoflagellates were most abundant, where as the highest contribution to biomass came from ciliates
It was found that heterotrophic protists consumed a mean average of 68.2% of chlorophyll a production, and that as the phytoplankton biomass increased, grazing rates also increased. small heterotrophic dinoflagellates were most strongly correlated with phytoplankton biomass than any other type of protist. These results correlate with results obtained from similar studies elsewhere in the world.

In waters with of a low phytoplankton biomass, the biomass relationship between phytoplankton and heterotrophic protists was found to be nearly equal, showing a significant reservoir of biogenic carbon represented by heterotrophic protists.

To conclude, the results obtained from this study further back up the idea that heterotrophic protists are the major consumers of primary production in the marine environment. It also reveals that grazing of heterotrophic protists on phytoplankton is one of the most important losses affecting biomass in the northern ECS.

The influence of coastal waters on distributions of heterotrophic protists in the northern East China Sea, and the impact of protist grazing on phytoplankton

1. Keun-Hyung Choi1, 
2. Eun Jin Yang2,*, 
3. Dongseon Kim1, 
4. Hyung-Ku Kang1,
5. Jae Hoon Noh1 and 
6. Cheol-Ho Kim1

J. Plankton Res. (2012) 34(10): 886-904 first published online June 28, 2012
http://plankt.oxfordjournals.org/content/34/10/886.full.pdf+html

Nitrogen fixation and transfer in open ocean diatom cyanobacterial symbioses

Nitrogen fixation and transfer in open ocean diatom cyanobacterial symbioses

There are a large proportion of diatoms that occupy ocean waters of a low nutrient level. As they are unable to acquire N from N₂ and the extracellular dissolved fixed inorganic nitrogen pools in the ocean are at such low concentrations, these diatoms are believed to form symbiotic relationships with N₂ fixing cyanobacteria, which transfer N to the associating diatom. There has been much controversy concerning the global N sources and sinks and it is believed the lack of research into such symbiotic relationships between cyanobacteria and diatoms may have resulted in a large underestimation of oceanic N₂ fixing rates.

Despite such symbioses being observed over a wide range of ocean basins there have been few previous studies concerning the N₂ fixation rate for such populations. This study utilised the technological advancements of NanoSIMS, high resolution nanometer scale secondary ion mass spectrometry, which enabled them to provide evidence of the N2 fixation and N transfer between individual cells. They used bulk samples taken from two regions of the Pacific Ocean which they incubated with ¹⁵N₂ in order to trace and quantify N₂ fixation. The observations involved the diatoms Hemiaulus, Chaetoceros and Climacodium, which associate with the cyanobacteria Richelia, Calothrix and Climacodium respectively.

The first experiments used epifluorescence microscopy to calculate the ¹⁵N/¹⁴N ratios and demonstrated that the enrichment pattern of Hemiaulus cells corresponded with the location of the Richelia trichomes. Furthermore, the chloroplasts of the diatoms were shown to be enriched, suggesting transfer of N to the diatoms. Similar results were also observed between the other associated cyanobacteria and diatoms being studied. Although N transfer has been observed via trichome from the cyanobacteria to the diatom, it is still unknown how efficient this exchange is as the symbionts remain outside the cell membrane of the diatom.

Since the diatoms inhabit oligotrophic waters it was believed they would have a slow growth rate and therefore only a small amount of N was expected to be transferred. Nonetheless, the experiments demonstrated enrichment in Hemiaulus cells after just 30 minutes and N saturation after 3 hours, providing further evidence that N₂ fixation rates may be greatly underestimated.

The N₂ fixation rates, transfer of N and growth rates all seemed to be accelerated under symbiotic conditions, therefore it was suggested the symbionts may be fixing a greater amount of N than their growth required. Given that the ¹⁵N/¹⁴N ratios estimated similar growth rates for all the symbionts and their corresponding diatoms it was predicted that Richelia fixed 71-651% more N than needed to support their growth. Considering it is energetically expensive to fix N₂ this suggests the diatoms may be influencing the N metabolism of their associated symbionts.

Even though there have been difficulties in estimating the densities of such symbioses, this study proves that fixed N₂ is directly transferred to associating diatoms, providing evidence that these populations may be an important underestimated source of N and therefore supports the argument that future models of global N should include such diatom symbioses. 

Nitrogen fixation and transfer in open ocean diatom cyanobacterial symbioses

Rachel A Foster^1,2, Marcel M M Kuypers², Tomas Vagner², Ryan W Paerl¹, Niculina Musat² and Jonathan P Zehr¹

The ISME Journal (2011) 5, 1484–1493; doi:10.1038/ismej.2011.26; published online 31 March 2011

http://www.nature.com/ismej/journal/v5/n9/full/ismej201126a.html#tbl1

Evidence of Phototrophy in Vibrio sp. by Expression of Proteorhodopsin: Starvation Response

Evidence of Phototrophy in Vibrio sp. by Expression of Proteorhodopsin: Starvation Response

The class of proteins known as the rhodopsins are the simplest light harvesting pigments, originally isolated from Archea as bacteriorhodopsin. These membrane embedded proteins produce an electrochemical gradient across the membrane when activated by light, where proton a proton gradient drives ATP synthesis. The discovery of proteorhodopsin among planktonic bacteria lead to the isolation of the gene cluster from the gammeoproteobacteria linage SAR 86 and transfer to heterologous bacteria E.coli to reveal light driven phototrophy similar to that of the Archeal protein bacterial rhodopsin. Further studies revealed the ubiquitous presence of proteorhodopsin among other marine bacteria, now thought to be present in 13-80% of bacteria. An inability to culture many marine bacteria meant that functioning of proteorhdopsin was only experimentally possible in E.coli until whole-genome-sequencing revealed the presence of proteorhodopsin in several culturable marine bacteria, including a Vibrio.

The is great significance to this investigation by Gomez-Consarnau et al (2010). Previously Vibrio’s have been described as organoheterotrophs, gaining nutrition from detritus or as opportunistic parasites. This investigation describes the functioning of proteorhodopsin in-situ by several mechanisms showing that vibrio’s enhance their survivability during periods of starvation by the expression of proteorhodopsin induced by light. Phylogenetic analysis has suggested that the lateral transfer of genes may be responsible for the presence of proteorhodopsin in Vibrio ADN4. It is also suggested that the presence of Leucine at position 105 modifies the the proteorhodopsin protein to absorb light maximally in the green part of the electromagnetic spectrum.

Gomez-Consarneu and colleges first measured growth and survival of  Vibrio Strain AND4 in light and dark regimes. No difference in cell yields was observed between light and ark regimes grown on a rich media. When AND4, grown on rich media, was transferred to natural seawater with low concentrations of nutrients reductive division was observed, characteristic of the starvation response in vibrio’s. The important value is that after 10 days starvation the culture in light was 2.5x higher than the culture in the dark.  

The optical densities were also measured in differing light regimes. In the dark the OD dropped off rapidly, but in the light the decrease was much less pronounced. After 7-13 days after starvation OD was 40-60% higher in light compared to dark. Further investigation took place to determine if it was light that was inducing the enhanced survivability of AND4 by producing a mutant deficient in the proteorhodopsin gene cluster by in-frame deletion. In line with prediction, the deficient mutant showed no significant survivability compared to control groups. Recovery time was also investigated with starved bacteria grown on rich-media. The results indicated that the wild-type AND4 grew 3-6 fold  faster after 5 day t compaerd the dark incubated bacteria.

This investigation adds a new dimension to our understanding of phototrophy in the ocean and what we thought we knew about the Vibrios. The functioning of proteorhodopsin was also been investigated by Wang et al (2012) in Vibrio campbellii , interestingly the results showed that continuous illumination actually decreased bacterial yields which the author suggests could be the result of photodegradation of cellular components or, the activation of lytic phase of embedded in the genome. Another possibility for the differences observed in cell growth under illumination between Gomez-Conarneu et al (2010) and Wang et al (2012) could be the luminescence used in the experiments. Gomez-Conarneu et al (2010) used luminescence values for continuous light of , 133 and 150 µmol photons m⁻² s⁻¹ whereas the luminescence used by Wang et al (2012) was considerable lower 42 µmol photons s⁻¹ m⁻².  

Gomez-Consarnau. L, Neelam.  A, Kristoffer. L, Pederson. A, Neutez. R, Milton. D. L, Gonzalez J. M, Pinhasi. J., (2012). Proteorhodopsin Phototrophy Promotes Survival of Marine Bacteria During Starvation. PLOoS Biol 8(4): e1000358. doi:10.1371/journal.pbio.1000358.  

http://www.plosbiology.org/article/info%3Adoi%2F10.1371%2Fjournal.pbio.1000358

Wang Z, O'Shaughnessy TJ, Soto CM, Rahbar AM, Robertson KL, et al. (2012) Function and Regulation of Vibrio campbellii Proteorhodopsin: Acquired Phototrophy in a Classical Organoheterotroph. PLoS ONE 7(6): e38749. doi:10.1371/journal.pone.0038749

http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0038749

Natural Variability in Coral Microbiotas: Has it Undermined our Understanding of Coral Health and Disease?

Investigations into the diversity of coral-associated microbes and inferences as to their roles within the coral holobiont (i.e. the coral host plus mutualistic Symbiodinium, bacteria, archaea, and fungi), have been based around the premise that coral microbiotas are important to the health and function of the host, and that alterations in this community are linked to coral disease.  However, without an understanding of the natural variability of coral-microbial communities and a realistic baseline description of healthy coral microbiotas, conclusions as to the causes and effects of coral disease are unlikely to be reliable.  Coral disease is a major threat to the future of reef-building tropical corals; with corals in chronic global decline, resolution of this issue could not be more urgent or relevant.

Recently, Kvennefors et al (2010) reported on the variability of the microbial communities associated with both healthy and diseased coral colonies: culture independent methods such as DGGE (of the V3 region of 16S rDNA) and FISH were employed to investigate the diversity and community structure of microbes associated with two coral species, Acropora hyacinthus and Stylophora pistillata.  Replicate samples were collected from three different sites around Heron Island (southern GBR, Australia), from both apparently healthy coral colonies, and those suffering from White Syndrome (WS).  The authors observed both species-specificity and site-specificity in the microbial communities of coral colonies, plus pronounced community shift in those affected by WS.  However, variability between replicates was also noted: a set of discrete coral-community profiles were described.  Species accumulation curves indicated that the minimum number of replicate samples needed to adequately investigate bacterial diversity was six (each collected from a different colony); Kvennefors et al (2010) concluded that at least six replicates were therefore needed to infer any influence of host species, location or disease.

The importance of this finding is illustrated by an investigation undertaken by Littman et al (2009): diversity of coral-associated microbes in three acroporid coral species was reported as varying between two GBR sites, but not between the host species, in healthy colonies.  Evidence of coral species specificity in associated microbial communities has been reported by many (e.g. Frias-Lopez et al., 2002; Bourne & Munn, 2005); however, it was suggested by the authors that lack of species-specificity in the microbiotas of these corals was due to the three host species surveyed being very closely related (in the same genus).   Littman et al (2009) collected three replicates of each coral species (in each site): Is the reported similarity in the microbiotas of closely related host species genuine, or would more extensive sampling suggest otherwise?  The evidence from Kvennefors et al (2010) indicates that without more adequate sampling, this question remains.

Possibly due to the time and money required to undertake the various molecular methods employed in such studies, work continues to be reported as having been undertaken using considerably less than the minimum number of replicates suggested by Kvennefors et al (2010).  This year (2012), Ceh et al published an investigation into the potential changes to coral-microbiota diversity and structure before and after a coral mass spawning event: two colonies per coral species were sampled.  It appears that under-replication may have inhibited our ability to understand the health (and thus disease) of the coral holobiont in the past; if so, it may continue to hinder us for some time to come.

 

Kvennefors, E.C.E. et al., (2010). Bacterial Communities of Two Ubiquitous Great Barrier Reef Corals Reveals Both Site- and Species-Specificity of Common Bacterial Associates. PLoS ONE 5, p.14

http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0010401

Littman, R.A. et al., 2009. Diversities of coral-associated bacteria differ with location, but not species, for three acroporid corals on the Great Barrier Reef. Fems Micriobiology Ecology 68, p.152-163

http://onlinelibrary.wiley.com/doi/10.1111/j.1574-6941.2009.00666.x/abstract

Ceh, J. et al., 2012. Coral-bacterial communities before and after a coral mass spawning event on iingaloo Reef. PLoS ONE, 7(5), p.e36920

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3353996/

(note: you may need to be logged into uni site for access to some papers) 

2005: A Bad Year To Be A Caribbean Coral

Coral bleaching is a well described anomaly in great reef ecosystems around the world. It has been described as the loss of intracellular endosymbionts, known as zooxanthellae, through expulsion. Zooxanthellae give coral reefs their high colouration and are arguably responsible for the beauty of the coral reefs. Once a coral has expelled its zooxanthellae it becomes a lighter or completely white appearance, hence the term "bleached". Coral bleaching can be caused by several stimuli, most prominently these include environmental triggers such as water temperature change, change in solar radiation received, changes in water chemistry and bacterial infection.

Mass Bleaching Events, where 100 km squared or more of coral is bleached, have been reported to have occurred on many occasions around the world in the last 30 years. However the most prominent of these occurred in 2005 in the Caribbean Sea. Whilst still under research, this event is reportedly caused by an anomalously warm water level and high surface light intensity from the months of June to October.

The authors of this paper use several methods to detect sea temperature variations. Most prominently these temperatures were detected by "satellite-based sea surface temperature" observations (SST), but also through manual methods which were subsequently checked with the corresponding location or pixcel from the satillite image. The sea temperature was reportedly increased by +1.2° C on average, with some areas peaking at +16° C. This elevated temperature lasted for many weeks and caused a 50% mortality rate in all Caribbean regions inhabited by coral. This is the worst case of thermal stress related mortality to date (however as mortality cannot be directly linked to the warm water anomaly, it is easy to critically disagree with the authors on this statement).

The increased temperature of seawater can lead to a loss of resistance to pathogenic disease by the coral, and an increase in the abundance of microbial pathogens in the surrounding waters. The authors speculate that opportunistic diseases can easily arise in this type of situation and more extensively bleached corals are immunocompromised. This is backed up by the strong correlation between disease outbreak and bleached corals.

Hurricanes passing within 100 km of coral reefs serve to call surrounding water and prevent this type of event, however in the year of 2005 several hurricanes passed too close to coral reefs and in addition to the long-lasting warmer water caused long-term damage to coral reefs that has lasted decades. When comparing this long-lasting damage to the remarkable stability of coral reefs during the prior 22,000 years, the authors conclude that warm water anomalies are human-caused but that the direct cause is unknown. However it is easy to dismiss this statement as no references or evidence is given to back this up.

Eakin C.M. et al (2010) Caribbean corals in crisis: record thermal stress, bleaching, and mortality in 2005, PLoS One, Published Online. Available from [http://hubs.plos.org/web/biodiversity/article/10.1371/journal.pone.0013969]

Are giant viruses gene thieves?

Giant virus with a remarkable complement of genes infects marine zooplankton.

The predominant theory about the origin of giant viruses is that they have acquired most of their DNA from horizontal gene transfer with cellular organisms, however little research has actually been done into the genomes of giant viruses.
Fischer et al (2010) look at the DNA and function of CroV, a giant virus which infects the bacterivorous marine flagellate Cafeteria roenbergensis. Only one other giant virus had previously been studied at this detail, and this was a freshwater Mimivirus.
As well as mapping out the genome of CroV the researchers used fluorescent in situ hybridisation to see which genes were expressed during infection which would show which genes were functional.
Although they found that half of CroV’s genes were similar to those previously found in eukaryotes, bacteria, archaea and other giant viruses, this paper shows the importance of studying the genetics of viruses alongside their hosts, as they could not verify that eukaryotic genes found in the virus were from C. roenbergensis due to the lack of knowledge of the host genome.
Fischer et al did find that CroV is far less dependent on host cell components than smaller viruses, and has the ability to make its own tRNA and tRNA modifying enzymes. Most of the genes atypical to viruses were expressed during infection suggesting that these genes were not simply non- functional DNA from horizontal gene transfer with cellular organisms, and this virus undertakes its own processes rather than relying on the host more than other viruses.
CroV also has DNA repair genes thought to be an adaptation to the high radiation surface waters where the host lives. These included DNA repair genes primarily found in bacteria and euryarchaeotes, but thought to have been acquired from another giant virus as it is most similar to those found in the Mimivirus previously studied. Also found were regions of DNA relating to glycoprotein biosynthesis that may be needed to create a protective outer coating to the virus.
Although this paper does not answer the question of whether CroV acquired most of its huge genome from horizontal gene transfer, the impression is given that the author thinks it did not. As much of CroV's DNA is more similar to the Mimivirus previously studied than it's host, the author believes that much of CroV's DNA is viral in origin, existing in an ancestor before the line had contact with eukaryotes.
Even though it does not answer the question directly, this paper may have brought us closer to understanding the origin of viruses and transposons in eukaryotes, as more recent work done on this virus revealed the Mavirus virophage which can only survive in the presence of CroV and showed that the origin of certain types of transposons is most likely viral.
The gaps in this study such as comparisons between the virus and host genomes, the function of repetitive DNA and ubiquitin in viruses, and a more in-depth analysis of the processes occurring during infection, would make interesting further work into this and other giant viruses. Further research on the ecological relationships this giant virus may have with its host and its parasite, and how the population dynamics of one would effect the other two would also be facinating, as this is only the second known example of a virus parasitising another virus.

Fischer M.G., Allen M.J., Wilson W.H., Suttle C.A. (2010) Giant virus with a remarkable complement of genes infects marine zooplankton. Proc. Natl. Acad. Sci. U.S.A. 107(19508)
http://www.pnas.org/content/early/2010/10/15/1007615107

Fischer M.G., Suttle C.A. (2011) A virophage at the origin of large DNA transposons. Science 332(6026):231–234.

Bioluminescence: What is  its role to marine snow? 

Bacteria use a variety of mechanisms to inhabit a large diversity of environments. One of these organisms is the bioluminescent bacteria which have the ability to produce light via quorum sensing. These organisms occur in a large diversity of environments including marine snow and in the photophores of fish. It has long been hypothesised that the bacteria living on the snow glow to mark the presence of a food particle for other organisms in order to get into their guts. This is termed the bait hypothesis. So far, this hypothesis has only been supported by showing that bacteria survive passage through fish guts. Once in the gut of fish, bioluminescent bacteria gain a nutritious environment for growth and a faster moving vector for dispersal.

 The objective of this study was to test the proposed steps of the bait hypothesis:

1.        The visual attraction of zooplankton to bacterial bioluminescence

2.       Promotion of glow in zooplankton ingesting bacteria (using planktonic brine shrimps as a surrogate for zooplankton)

3.        Attraction of fish to glowing prey

4.        Survival of bacteria in the guts of both zooplankton and fish.

Zarubin's experiment was set up in a sea water tank, this was used to examine if the luminescence of Photobacterium leiognathi attracts Decapod, Mysid and Copepod zooplankton. Zarubin et al observed significant changes in the distribution of Decapods and Mysids but not in Copepods. The brine shrimp Artemia salina (the surrogate to test the promotion of glow) became luminescent after swimming in a culture of P. leiognathi. Using long-exposure photographs, the luminescence in the guts of Artemia were clearly visible, with additional glow produced by bacteria attached externally. Similarly, non glowing individual marine Mysids, Anisomysis marisrubri started to glow after contacting P. leiognathi.

Zarubin and co found that glowing A. salina increased risk of predation by the fish Apogon annularis in dark. Almost all glowing Artemia were consumed compared with rare predation of non glowing specimens, the authors stated that this occurred only when the prey drifted by chance directly toward  the predator. Glow was detected in faeces of Artemia that fed on the bacteria which indicated that the luminescent bacteria survived the passage through the guts.

This study provides experimental evidence for some steps of the bait hypothesis. But the author’s did not differentiate between external and internal glow on the surrogates. The authors argue that both sources occur in nature, but cells attached externally are not following the proposed steps of the bait hypothesis. Therefore these results should not be used to answer steps 1 and 2 of the bait hypothesis in my opinoun. Zarubin et al used Artemia as a surrogate for zooplankton prey in this experiment because it is readily eaten by fishes, easy to handle, and lacks evasive behaviour. But I do not agree with this choice of organism because it is a freshwater organism that is being used as a model for an experiment which relates to deep sea ecology and therefore is not an appropriate choice. The authors argue that the luminesce observed in A. marisrubri had the same intensity as Artemia but they have not made it clear why A. marisrubri was not chosen in the first place. Although the experiments with P. leiognathi did not fully simulate in situ conditions I think this study provides a good basis for experimental testing of the bait hypothesis. However, experiments with real marine snow are needed to test this claim explicitly.

 

I chose to review this study due to my interest in bioluminescent bacteria. I also have a large curiosity in  marine snow ecology and how these bacteria are affecting it.

 

http://www.pnas.org/content/109/3/853.full