Oysters with a side dish of... Probiotic bacteria?

Paralytic shellfish toxins (PSTs) are non-protein neurotoxins produced by saltwater dinoflagellates and freshwater cyanobacteria. They are a group of water-soluble carbamate alkaloids, which are either non-sulfated (saxitoxin (STX), neo-STX), singly-sulfated (gonyautoxins (GTX)) or doubly-sulfated (C-toxins). A high intake of PST leads to paralytic shellfish poisoning, where the toxins block the influx of sodium channels, restricting signal transmission along neurons therefore causing death from respiratory failure. The effect of long-term, low level exposure to PSTs is unknown, however most cases where humans are affected occur through contaminated seafood (such as mussels and oysters), algal dietary supplements and toxin-producing cyanobacterial cells. Human paralytic poisoning has been an increasing problem in the food industry, where seafood regulations set the maximum acceptable limit of PSTs in shellfish at 80 µg STC equivalents 100g^-1 tissue. There is no known antidote or cure for paralytic shellfish poisoning, and most methods involving removing PSTs have limitations. 

This study by Vasama et al. (2014) looks at using two probiotic lactic acid bacteria, Lactobacillus rhamnosus strains GG (GG) and LC-705 (LC-705) (in viable and non-viable form), in removing six PSTs from acidic and neutral solutions, mimicking the pH variation in the gastrointestinal tract. Both bacterial strains (GG and LC-705) in both viable and non-viable forms were cultured, and cyanobacterial extract containing PSTs (taken from a bloom of toxic Anabaena circinalis) were obtained and prepared to give two pH level solutions. Pure PST solution was prepared and each strain of bacteria was suspended in either Pure PST solution (as a control) or one of the two pH PST solutions (pH 7.3 or 2.0). High Performance liquid chromatography was then performed on the samples to detect each of the three classes of PSTs (STXs, GTXs and C-toxins). 

The results showed a strong removal of PSTs by non-viable bacteria indicating that PSTs are possibly removed by binding rather than metabolism. The highest degree of removal was observed for STX and neoSTX (77%-97.2%). The effect of non-viable and viable showed no significant difference therefore suggesting viable bacteria may also remove PSTs through binding, which is consistent with previous reports that specific bacterial strains remove a range of mycotoxins through binding. 

Overall, our knowledge on absorption of PSTs through mammalian intestinal epithelium is still limited, however this is the first study to show that by possibly altering the intestinal microflora composition using probiotics, the uptake of harmful compounds by the body when ingesting PSTs can be decreased and symptoms of sickness can be prevented. This can therefore lead to development for industrial applications or health benefits. 

Vasama, M., Kumar, H., Salminen, S. & Haskard, C. A., 2014. Removal of Paralytic Shellfish Toxins by Probiotic Lactic Acid Bacteria. Toxins, Volume 6, pp. 2172-2136. 

http://www.mdpi.com/2072-6651/6/7/2127/htm

Sick of Quorum Sensing? So are Corals (…maybe)

It’s no secret that coral reef health worldwide is on the decline. A significant contributing factor to this is the increase in incidences and severity of epizootics in the last 30 years. These diseases have the potential to decimate coral populations and yet little is known of their etiology and ecology. White band disease (WBD) for instance, a phenotype prevalent throughout the Caribbean, has destroyed 95% of regional Acropora populations since 1979 and yet many of the virulence factors that cause it are still poorly understood.

White band disease typifies most coral diseases in that it is characterized by tissue degradation along a coral branch. The disease, among many others, is thought to be the result of opportunistic pathogenesis by existing coral holobiont microbiota and so in a study by Rebecca Certner and Steven Vollmer the role of quorum sensing in WBD associated microbes was examined in order to further understand the etiological factors driving the infection.

The investigation of quorum sensing mechanisms was achieved by inoculating both healthy and diseased corals with cell-free culture fluid from healthy or diseased to coral homogenates to examine the resulting effect on the transmission and progression of WBD. In addition to this N-Hexanoyl-DL-homserine (AHL), a common autoinducer used by virulence-associated microbes, was added to coral samples and the resulting shift in host microbial composition examined to assess whether the AHL would induce or amplify WBD-like symptoms.

Interestingly the results showed that CFCF from diseased corals appear to cause significantly greater yield of Cytophaga-Flavobacteria in both healthy and diseased samples which would suggest an induction of WBD-like shift in community composition due to the CFCF addition Cytophaga-Flavobacteria. Healthy CFCF however appears to inhibit Cytophaga-Flavobacteria growth which implies it may contain antimicrobial molecules, a phenomenon often referred to as coral-probiotic hypothesis. In fact, because Cytophaga-Flavobacteria are known opportunistic pathogens the result appears to corroborate the compromised-host hypothesis as it implies that the disease symptoms may be induced by increased vulnerability of the coral host that allows such potential pathogens to outcompete commensal microbes.

The addition of AHL meanwhile, resulted in tissue loss and total mortality of all samples, both healthy and diseased, whereas the healthy control sample remained healthy and alive for the duration of the study. This strongly evidences that AHL plays a pivotal role in disease virulence. This acyl-homoserine lactone quorum sensing system is well characterised in many suspected marine pathogens from the Vibrio genus but has not previously been demonstrated in coral-associated Cytophaga-Flavobacterium. In many Vibrios virulence is positively and negatively controlled by quorum sensing in order to regulate virulence-associated genes and so it is likely that this system is also involved in the repression of host immunity. It is possible therefore that such a ability to disrupt host immunity is also present in Cytophaga-Flavobacteria.

Overall this study provides a vital insight to the mechanisms via which disease-causing agents are able to influence holobiont microbial population. It is obvious that N-Hexanoyl-DL-homserine has a significant influence on the growth of coral-associated bacteria and therefore its presence around coral hosts could act as an early warning for the pathogenesis and infection of diseases.

Certner, R. H. and Vollmer, S. V.. (2015). Evidence for Autoinduction and Quorum Sensing in White Band Disease-Causing Microbes on Acropora cervicornis. Scientific Reports. 5, 11134.

Bacterial plasmids may show the origin of TTX

Tetrodotoxin (TTX) is a highly toxic neurotoxin found in a diverse range of genetically distant species. It has been proposed that bacteria are the true source of TTX within animals, such as pufferfish, gastropod, octopus etc.; however it is difficult to truly isolate this toxin’s source.

A study by Liu et al. (2015) wanted to identify the origin of TTX within cells of Aeromonas sp. Ne-1 isolated from the pufferfish, Takifugu obscurus. This was completed using the plasmid involved in TTX production, pNe-1. It was found that this plasmid’s copy number was associated with the bacteria’s ability to produce TTX; when the copy number decreased, so did the concentration of TTX. Unexpectedly, a degradation enzyme was discovered within the cells, which may explain why the energetically costly production of TTX reduced over time.

The plasmid of Aeromonas sp. Ne-1 was extracted and sequenced in order to determine the open reading frames (ORFs) present. At least 60 ORFs were recorded, with one known to be associated with E. coli insertion elements, and another associated with the incorporation of DNA into a larger DNA molecule. These potential genes could facilitate the transfer of the TTX pathway horizontally between organisms, which may explain the presence of TTX in over 14 different phyla.

A time-course bacterial cell culture was grown and at 12, 18, 21, 24, 31 and 42 hours samples were taken for plasmid extraction. The plasmid DNA was used in real-time qPCR to determine the average plasmid copy number. From 12 to 18 hours, there was an increase in the plasmid copy number, as expected. What is interesting is that after the 18^th hour, the copy numbers of this plasmid decreased sharply from 2.51 to 0.27, and eventually the plasmid was undetectable at 42 hours.

Another time-course cell culture was set up in order to measure the TTX concentrations at 24, 42, 66 and 96 hours. The results were similar to above, as after 42 hours (1.602 ng for each 10¹⁰ clone), the concentration dropped sharply; at 66 hours the concentration fell to 0.292 ng, and then down to 0.220 ng for each 10¹⁰ clone at 96 hours. This decrease in TTX concentration suggests an unknown TTX-degrading enzyme in the cell. A follow up experiment was conducted to determine the presence of such an enzyme, whereby a set amount of TTX was added to cultures. Compared to the control, at 66 and 96 hours there was 26-55% and 53-69% degradation of TTX, respectively.

This paper has shown how the plasmid copy number of pNE-1 from Aeromonas sp. Ne-1 is related to TTX production, and that there is a high possibility that the genes involved in this pathway are passed from each organism via horizontal gene transfer (HGT). What was unexpected was the presence of a TTX-degrading enzyme, which suggests that there is a homeostatic control mechanism within the bacteria that maintains stable levels of the energetically costly toxin.  

I believe that this work has furthered our understanding of the potential origins of this prevalent toxin, despite the widespread distribution across many phyla. For the first time, it has been seen that the bacteria Aeromonas sp. Ne-1 can produce and degrade the levels of TTX in order to control their internal environment. This along with the possible origins of this toxin being spread via HGT is a massive breakthrough in understanding how this pathway may have arrived in many animals today and what may regulate it.

Liu, J., Wei, F., Lu, Y., Ma, T., Zhao, J., Gong, X., & Bao, B. (2015). Production level of tetrodotoxin in Aeromonas is associated with the copy number of a plasmid. Toxicon, 101, 27-34.

http://www.sciencedirect.com/science/article/pii/S0041010115001063

A helpful website explaining some plasmid characteristics:

http://blog.addgene.org/plasmid-101-origin-of-replication

Effects of increased temperature on DMS(P) concentration in coral associated Symbiodinium

Worldwide coral reefs face climate change. Recent events have shown that changes in coral reef ecosystems can lead to disease such as bleaching. Anyhow, coral reefs have existed for hundreds of thousands of years and have survived climate change events before. To understand how coral reefs respond or even adapt to climate change, it is necessary to have a closer look on the small things in life of a coral.

Most reef building corals are constructed trough the association between coral polyps and their symbiotic dinoflagellate microalgae. Besides providing the host with fixed carbon, the zooxanthellae (genus Symbiodinium) also contribute to the production of antioxidants to prevent oxidative damage. As part of the coral holobiont zooxanthellae produce and process dimethylsulphoniopropionate (DMSP) and its enzymatic cleavage product dimethylsulphide (DMS).

DMSP and DMS play a key role in the global sulphur cycle and also in the antioxidant system of the coral.

Depending on their symbiotic zooxanthaellae corals respond differently to climate change. Some clades of zooxanthallea are known to be more resistant to e.g. temperature changes. Increasing temperature is one of the main factors for coral bleaching.

The study by Deschaseeaux et al. (2014) investigated the tolerance of the symbiotic zooxanthellae to increasing temperature linked to the production of DMSP and DMS. Therefore, two different subclades of symbiotic algae (Acropora millepora (D1) and Acropora tenuis (C1)) were collected in waters of Magnetic Island, Australia. Clade D is known to be more resistant to thermal stress than clade C.

The two clades were isolated from the coral tissue. To prevent contamination the zooxanthellae were kept in an axenic chamber. The genotypes of the clades were identified. Clades were examined in chambers with increasing temperature and in control chambers. DMSP and dissolved and gaseous DMS were measured.

Relevant results were that DMSP and DMS concentration differed between the two clades. DMSP concentration decreased in all treatments. Although, concentration of DMSP were higher in clade C than in D. No difference in DMSP concentration was measured between the two treatments of clade D. For clade C the concentration of DMSP was higher in the control chamber than in the chamber with increased temperature. The higher concentration of DMSP in the control chamber might be a result of the enhanced DMSP consumption in clade C with increasing temperature. Suggesting that clade C uses DMSP to prevent oxidative stress. This is supported by the results of measured DMS. Decreasing concentration of DMSP didn’t lead to increasing concentration of gaseous DMS in clade C. Potentially caused by consumption of DMS under thermal stress. All in all, clade C showed distinct response to thermal stress, whereas clade D remained nearly unaffected.

Despite of some vague results, this study contributes to a better understanding of coral reef response to increasing temperature. Considering the theory that coral produced DMS plays a role in cloud forming and therefore has an impact on local climate, enhanced consumption of DMS(P) due to thermal stress by clade C could affect this interaction. Secondly, it can be assumed that increasing temperature has distinct effects on the biogenic sulphur cycle of symbiotic zooxanthellae.

Furthermore, this study focused only on the DMS(P) production by symbiotic zooxanthellae. The whole microbial holobiont should be considered for further studies.

Additionally, I would like to recommend this paper by Nils Rädecker, a master student of the Centre for Tropical Marine Ecology, Bremen. The paper gives you a nice overview of nitrogen cycling in corals. Even with a short video at the end: http://www.sciencedirect.com/science/article/pii/S0966842X1500075X

E.S.M. Deschaseaux, V.H. Beltran, G.B. Jones, M. A. Deseo, H.B. Swan, P.L. Harrison, B.D. Eyre (2014) Comparative response of DMS and DMSP concentrations in Symbiodinium clades C1 and D1 under thermal stress. Doi: 10.1016/j.jembe.2014.05.018. http://www.sciencedirect.com/science/article/pii/S0022098114001373

Stony corals acidify their zooxanthellae

Corals are marine organisms that are well known for their symbiotic relationship with a genus of dinoflagellate algae called Symbiodinium (better known as zooxanthellae). The coral provides the zooxanthellae with dissolved inorganic carbon along with other growth factors and the zooxanthellae provides the coral with organic carbon, factors for coral skeletal growth and even a buffering system for when the coral lays down calcareous material. The dinoflagellate stays inside the coral's inner tissue in a membrane-bound space called the symbiosome. It is generally agreed that these corals will be harmed by ocean acidification, however a study by Barott et al. gives evidence that certain corals subject their zooxanthellae to acidified conditions during their symbiotic life cycle. 

Two species of Scleractinian corals, Stylophora pistilata and Acropora yongei were investigated in the study . By using epifluorescent microscopy of coral tissue with intact symbiosomes and symbionts, the researchers visualised proton pumps called VHAs lining symbiosome membranes inside coral cells. These VHAs transport protons into the lumen, creating acidic conditions around the algae. Using Lysosensor Green (LSG), a dye that fluoresces brighter at higher pH, the researchers were able to determine the pH in the various tissues. In the corals tested, the symbiosome was around pH 4, although some areas tested were as low as pH 2. This may be to shift the CO_2 <---> HCO₃^- (bicarbonate) equilibirium towards CO₂ to increase the rate of photosynthesis by the algae. 

Interestingly, both the coral and algal cytoplasm pH remained around 7. The paper didn’t detail how the coral controls its own neutral pH whilst also maintaining an acidic vacuole; this would be very interesting to look in terms of projected ocean acidification. If the corals can create membranes that withstand up to pH 2 and still seem to undergo nutrient transmission, it’s possible they could “armour” their ectoderm (outer membrane) with them to withstand higher ambient acidity. A thought on the other hand is that any flux of environmental acidity will disrupt the corals ability to maintain the different pHs in its tissues and damage them. 

This study claims to be the the first of its kind, and presents a very interesting findings indeed. Identifying a new process in the coral tissues provides the scientific community with a new factor to monitor coral health and investigate experimentally how projected climate change might influence this new process. A criticism for this paper is that in the results it uses “corals” a lot, giving the impression they are applying their results to “corals” as a whole. The study only used two species of one order of corals which -- whilst representative, perhaps, of the order— shouldn't be applied without further tests to higher or other taxa of coral. Whilst criticising the narrow scope of species studied I must also acknowledge this study sets groundwork for future tests on other coral groups.

Reference: 

Barott, K.L. Venn, A.A. Perez, S.O. Tambutté.S. and Tresguerres, M. (2015) Coral host cells acidify symbiotic algal microenvironment to promote photosynthesis. PNAS, 112 (2) 210-215.

Are corals fighting back?

Corals survive via a mutualistic and obligate symbiosis with dinoflagellate algae of the genus Symbiodinium. These symbionts, called zooxanthellae, provide their coral hosts with the majority of their carbon, and therefore enable the corals to survive, grow and ultimately, reproduce. Acquisition of these symbionts is generally done by one of two methods. Vertical transmission, means a specific zooxanthellae will be passed from the parent coral to their offspring. This saves the progeny of having to find and acquire their symbionts, but it also very limiting. If a coral planulae has acquired the same symbionts that its parent had, then it is likely to run into trouble if its environmental conditions change, as the symbiont is likely to be better suited to the parental environment than the new one. There is much less room for adaptation. The alternative is horizontal transmission, where coral planulae begin life with no zooxanthellae, and soon have to find and acquire their symbionts from their environment, allowing them to take up environmentally relevant symbionts, and therefore are often better able to adapt to the ever changing environment.

Byler et al. (2013), studied the coral species Stylophora pistillata, a coral which has shown it is able to acquire zooxanthellae via both mechanisms, a rare phenomenon. They studied individuals in the planula, juvenile and adult stage, from shallow and deep habitats, over a 3 year course, using PCR, DGGE and Real Time PCR to detect and identify both dominant and low-level symbionts within the coral samples.

Their results showed that not only can this species use both forms of acquisition, but that there is also a difference in its ability to do so based on the type of habitat it was taken from. To begin with, the adult stage and the planulae symbionts from each habitat type were identified. It was stated that while the shallow water corals only hosted A1 type symbionts (both the adult and the planulae), the deep water planulae only hosted C72 type symbionts initially, and later on, in their adult lives, also played host to low-level symbionts, such as those from clade A.

The most likely, and simplest explanation for this is that the corals are able to horizontally acquire other surrounding (free-living) symbionts somewhere between their planula and adult stages, as when adults which hosted more than one type of zooxanthellae created new offspring, their offspring were found to only host the most dominant (original) symbiont in their initial stage of life.

While subsequent vertical transmission of a symbiont which has been acquired horizontally has not been seen over the course of this experiment, that is not to say that it isn’t possible, as it is likely that it could take 100’s or 1000’s of years for this to evolve.

I feel this paper provides an interesting insight into an adaptive mechanism not previously thought to occur in the natural environment. While it is in no means a quick fix for corals under the threat of climate change, and not even an event that is applicable to all corals, it does show that we don’t yet fully understand even some of our most researched organisms. I believe it would be interesting to see exactly what advantages this multiple acquisition method gives the corals, and to what extent it works under changing conditions.

K. A. Byler, M. C.-V. (2013). Multiple Symbiont Acquisition Strategies as an Adaptive Mechanism in the Coral Stylophore pistillata. PLOS.

Available at:

http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0059596