Do temperate corals rely on their microbes to the same extent as their tropical cousins?

Tropical coral reefs have had a lot research investment to understand the complex relationship between eukaryotic host and the microbial community. It is generally accepted that tropical corals host a species-specific microbial community (Rohwer et al 2002: Bourne & Munn, 2005), which play vital roles in nutrient gathering/fixing in the oligotrophic waters, along with antibiotic production and niche filling to prevent disease (Rohwer et al, 2002).

Temperate corals have had much less attention and little is known about whether they harbour specific communities or why disease prevalence is becoming more apparent?

Here, the author has compared the microbial community of the temperate coral Eunicella verrocosa during summer and autumn, as well as between healthy and diseased states to give some indication to what roles microbes play in this species of temperate coral. Ransome et al (2014) used denaturing gradient gel electrophoresis (DGGE) and clone library constructions to build a ‘fingerprint’ of the community structure, and compared the community’s at different sites, season and health to see if the microbial community changed. DGGE is an effective semi quantitative method that is benefitted by being culture-independent, as culture dependent methods are often limited as few marine microbes are culturable.

Ransome et al (2014) found significant differences in the community composition between season, and between health states. It is also documented that a distinct community was found in the water column compared to the corals; however the water samples were taken eight miles from the coral site, and no coral were present at this site, meaning the water sample was not representative. It would be expected that the microbial community at a different location would be distinct from the coral community. For that reason I find it difficult to draw any conclusions from this comparison. I think the author is trying to illustrate that the coral community is often distinct from water column community, because the corals selects a beneficial community. This point was well documented in tropical corals by Bourne and Munn (2005) and Richie (2006).

The author documented a change microbial community, with increased diversity when diseased, similar to findings in tropical corals reported by Pantos et al (2003) & Cooney (2002).

During autumn the microbial community was more abundant, and disease occurred more frequently, autumn (n =6), summer (n=1). It is unknown what causes the increase in disease, but one mechanism might be increased microbial activity due to higher nutrients content and dissolved organic carbon during autumn, highlighted by Smith et al (2006).

The author used three study sites in this paper, which were very different to one another, with differences in light intensity, sedimentation and substratum type; without accounting for disease there was a significant difference in community composition between sites. I think this paper would have benefitted from having more similar sites to make any differences caused by season clearer.  Or to have had more repeats to address the variables: light, sedimentation or substratum, and not used time of year as a variable too.

This paper was the first paper to gain molecular data on the bacterial community associated with Eunicella verrocosa in healthy and diseased states, which suggested that Eunicella verrocosa might harbour a species-specific community. There was a different and more diverse community found on the diseased coral, which is also observed in tropical corals, indicating a possible similarity between temperate and tropical corals, in causative agents of disease.

Some interesting questions have arisen from this paper such as how does the microbial community change seasonally in this temperate coral, and is the prevalence of disease in autumn linked to increased temperature or due to higher nutrient content? If you were to consider the substantial differences in their environmental conditions, one might expect a substantial difference.  For example, in the tropics the nutrient content is always low and corals rely on the microbes to accumulate limiting nutrients. In temperate seas variations occur seasonally, with nutrient concentrations high in the winter and depleted in the summer. Their dependence on microbes may vary seasonally, related to the changes in nutrients. 

Primary Reference :

Ransome, E., Rowley, S. J., Thomas, S., Tait, K., & Munn, C. B. (2014). Disturbance to conserved bacterial communities in the cold‐water gorgonian coral Eunicella verrucosa. FEMS microbiology ecology.

 

http://onlinelibrary.wiley.com/doi/10.1111/1574-6941.12398/abstract

Other references:

Bourne, D. G., & Munn, C. B. (2005). Diversity of bacteria associated with the coral Pocillopora damicornis from the Great Barrier Reef. Environmental Microbiology, 7(8), 1162-1174.

Cooney, R. P., Pantos, O., Le Tissier, M. D., Barer, M. R., & Bythell, J. C. (2002). Characterization of the bacterial consortium associated with black band disease in coral using molecular microbiological techniques. Environmental Microbiology, 4(7), 401-413.

Pantos, O., Cooney, R. P., Le Tissier, M. D., Barer, M. R., O'Donnell, A. G., & Bythell, J. C. (2003). The bacterial ecology of a plague‐like disease affecting the Caribbean coral Montastrea annularis. Environmental Microbiology, 5(5), 370-382.

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

Rohwer, F., Seguritan, V., Azam, F., & Knowlton, N. (2002). Diversity and distribution of coral-associated bacteria. Marine Ecology Progress Series, 243(1).

Smith, J. E., Shaw, M., Edwards, R. A., Obura, D., Pantos, O., Sala, E., ... & Rohwer, F. L. (2006). Indirect effects of algae on coral: algae‐mediated, microbe‐induced coral mortality. Ecology letters, 9(7), 835-845.

 

 

 

Vibrio cholerae also afraid of something and he run away.

- Maybe the deadline is over but I was working on this post so I posted it anyway, peace and love-

The ocean water is an heterogeneous environment where microorganisms must navigate to locate resource and conditions suitable for their growth. Chemotaxis is a fundamental mechanism that allow planktonic microorganism to aggregate around phytoplankton cell, colloidal particle, plume of organic substrates and resources. In such environment specific secondary metabolite can influence the behaviour of motile marine bacteria and a good example can be the quorum sensing for intraspecific signalling or antibiotic compounds for interspecific competition.

It’s knew that Vibrio SWAT3-wild-type (SWAT3-wt) and the human phatogen Vibrio cholerae are both particle-attached bacteria and the first one express antibiosis. SWAT3-wt can produce andrimid (inhibitor of acetyl-CoA carboxylase, an essential enzyme) that instead SWAT3-111 (a mutant) cannot produce. The andrimid potently inhibits the growth of V. cholerae but there are few informations on the effect of such molecule in much real natural condition as sub-lethal concentration.

V. cholerae is a particle attached bacterium and the authors in this work try to understand if andrimid can have a role in antagonistic interations with the other Vibrio species. They develop a chemotaxis assay combining a microchannel and a diffusion disk (on an agar surface Fig.1) to quantify the swimming behaviour, speed and turning rate. They used microvideography and cell tracking methods to monitor the swimming pattern of V. cholerae when exposed to lethal concentration of andrimid (i.e. cell stop swimming) and when exposed to sub-lethal concentration, either of the pure compound or when produced by a colony of SWAT3-wt.

To have a control reference, the authors performed two assays, one (1) with no chemical stimuli (to test the effect of the microchannel) and another (2) with a growing colony of SWAT3-111 (to be sure that other secondary metabolites don’t give effects). For both controls the swimming behaviour of V. cholerae were not related to the position in the microchannel and distance from the growing colony. They measured swimming speeds of 52.6 and 53.2 μm/s for (1) and (2) respectively. Then exposing V. cholerea in two andrimid gradients (one from pure antibiotics and one from a growing colony of SWAT3-wt) they found similar values of motility between controls and in a zone called non-lethal zone (1-1.2 cm distant from the source). In contrast, in a nearer zone to the sourse (called sub-lethal) they found significantly altered swimming behavior. V. cholerae was faster of about 25% (67.4-65.6 μm/s) in comparison to the controls. Also was defined a lethal zone where the bacteria showed a slower swimming speed (35% less than controls). Another interesting results were the turning rate and the run length (30% more) both increased in the sub-lethal zone meaning that V. cholerae run away from the danger. Were also measured swimming trajectories and the result was significantly shifted way 180° direction, so again away from the source of andrimid.

This results gave more information on species-specific competition for resources since the two Vibrio species tested here are both reported to colonize particulate organic matter in planktonic environments. The authors indeed suggest a mechanism to explain the observed decrease attachement by V. cholerae to particle previously colonized by SWAT3. So this rise some questions in my mind: how abbundant is SWAT3 in nature? May it explain also the fluctuation of V. cholerae outbreak in particular location? Such interaction can take place near nutrient rich particles likely determine the winner in the competition for space and resources and ultimately the abundance of V. cholerae in environment. Obviously more molecular study on this field could give additional data on this chemotaxis related competition. Maybe monitoring the abundance of SWAT3 in natural samples can also give some more light on the difficult manage of harmful outbreak of disease related to V. cholerae.

Graff, J. R., Forschner-Dancause, S. R., Menden-Deuer, S., Long, R. A., & Rowley, D. C. (2013). Vibrio cholerae exploits sub-lethal concentrations of a competitor-produced antibiotic to avoid toxic interactions. Frontiers in microbiology, 4.

The risks of Domoic acid poisoning in humans.

Domoic acid (DA) is a biotoxin produced by the marine diatom from the genera Pseudo-nitzschia.  It is known to cause amnesic shellfish poisoning in humans, it has neurological and gastrointestinal effects but may also result in seizure or coma.  It is ingested through consumption of shellfish, which are known to accumulate the toxin for longer periods of time than other fish or shellfish.  Previous studies on scallops have found the toxin to be present in levels exceeding the regulatory limit of 20 μg/kg in up to 17% of tested samples within the EU. 

Although there are measures in place to limit consumption by humans it is unknown what effect low levels of this toxin may have, if it will build up over time or even if it could be passed from mothers to their unborn children as has been seen in rats.  Although the tolerable daily intake (TDI) for humans is set at 0.075 ppm there have been no apparent clinical effects found for levels of less than 1.0 ppm.  That being said, other edible marine species such as razor clams and crabs have TDI’s of 19.4 and 31.5 ppm respectively.  Should these be accidentally consumed they could have dire consequences for humans.

A team in Belgium have analysed shellfish samples and data obtained from national food agencies to try to determine the actual risks to humans from eating scallops and oysters in particular. From their sample of shellfish (Oysters, Mussels and scallops), they discovered 22% of their scallops had DA levels higher than the regulatory limits, this was the highest percentage of all shellfish.  By extrapolating this data they calculated that actually less than 1% of the Belgian population would be at risk from amnesic shellfish poisoning due to eating shellfish with levels of DA that exceeded the acute dose. 

There are fairly stringent procedures in place to ensure acute exposure is limited however the effects of long term exposure are currently unknown.  It is also not known if cooking definitely reduces concentrations of DA in all species and this is certainly an area that could be fairly easily evaluated.  The study also highlighted the fact that long term exposure could actually affect as many as 5% of the population and confirmed that certain groups of the population such as pregnant women, young children or those with any form of reduced liver function could be at greater risk of poisoning. 

Given that DA poisoning can not only effect humans but also some marine mammals and birds it is certain that research into accumulation of DA and the way it may move through the food chain is essential.  There have been fairly recent advances in ways to detect the poison using enzyme-linked immunosorbent assay (ELISA), which now means that testing is much cheaper than it has been previously.  Currently there seem to be only isolated outbreaks amnesic shellfish poisoning but going forward it will be important to discover what effects environmental changes such as global warming may have on both the diatoms themselves and also on the species that accumulate the poison.  We must also consider the fact that as availability of fish populations changes shellfish may grow in popularity which would again increase the risk of outbreaks and isolated cases.

Andjelkovic, M., Vandevijvere, S., Van Klaveren, J., Van Oyen, H., & Van Loco, J. (2012). Exposure to domoic acid through shellfish consumption in Belgium. Environment international, 49, 115-119.

New digestive symbiosis in the hydrothermal vent amphipoda Ventiella sulfuris

New digestive symbiosis in the hydrothermal vent amphipoda Ventiella sulfuris

Ventiella sulfuris is the most abundant amphipod species inhabiting the Eastern Pacific Rise vent field, and is commonly found near population of the Pompeii worm (Alvinella pompejana)

Usually, species living on/near hydrothermal vents survive due to their symbiotic relationships, commonly with chemoautotrophic bacteria. These symbionts often aid their hosts by supplying them with nutrition, which they themselves obtain through sulphur or methane energy sources. For this reason the authors collected specimens of V. sulfuris with the objective of understanding and documenting the bacterial symbiosis occurring in the species (if it did occur), and to hypothesise how this amphipod gains nutrition.

Gut contents of V. sulfuris contained bacterial traces (empty walls or sheaths) and non-degraded Alvinella pompejana cuticle, suggesting that these amphipods feed on A. pompejana and/or their respective symbionts. A previously hypothesis suggested that V. sulfuris grazed directly on the epibiotic bacteria, found on the external surface of the Pompeii worm. This theory is supported by gut condense as they discovered no inner tissue from the worm, only surface tissue; alongside an abundance bacterial traces.

Light (LM) and electron microscopy (SEM and TEM) revealed the outer body, surface, gills and appendages of the host are free from micro-organisms; however the digestive system (the midgut and the hind gut) contains two major microbial communities which were observed in all organisms examined.

In amphipods the midgut and digestive gland are the major organs involved in digestion and absorption of nutrients. In this species electron microscopy revealed the posterior section of the midgut harboured a community of Gram-negative, single-cell, long rod-shaped bacteria. Some of these bacteria were in contact with the endodermal cell membrane, and high magnification revealed numerous membrane vesicles and debris between the bacterium. This is an unusual location, as this is a highly absorptive part of the digestive tract. Additionally, there was an absence of bacteria in the digestive gland.

In the hindgut, the authors observed that some cells exhibited numerous mitochondria that seemed larger than those found in other regions of the digestive system. Electron microscopy revealed densely packed epimural rod-shaped bacteria that (at high magnification) exhibited two morphotypes (electron-lucent short rods, and electron-dense thin-long rods). Both forms were tightly attached to the cuticle or citicular spines, and formed thick mats embedded in a dense organic matter.

16S rRNA gene diversity of all bacterial communities found in host were molecularly analysed to revealed 12 phylogenetic groups; the two most common of which included the Epsilonproteobacteria and the Firmicutes. Interestingly, many of the ID’ed bacterium were closely related to mammalian gut microflora. However some phyla, such as Epsilonproteobacteria are commonly associated with deep sea and/or hydrothermal vent symbiosis.

Sequencing analyses of 16S rRNA genes revealed that the bacteria found in the digestive system of V. sulfuris belong to six phyla: Epsilonproteobacteria, Firmicutes and Cytophaga– Flavobacter–Bacteroides, Gammaproteobacteria, Betaproteobacteria and Alphaproteobacteria. Most of these sequences are close to symbiotic bacteria of hydrothermal vent organisms, and bacteria involved in digestive symbiosis.

Due to a low sample size and an unsuitable fixation of specimens, bacterial DNA was partly degraded and therefore FISH (a technique that could have successfully correlated bacteria phylogenetic affiliation to their location) the authors were unable to specifically ID the two bacterial groups that were discovered in the gut. They did speculate that the midgut community was probably Epsilonproteobacteria, and the foregut phyla were likely to be Alphaproteobacteria, Firmicutes and/or CFB.

Overall an interesting study, but a real shame they couldn't pinpoint the exact phyla/species of the symbionts. However, they are one of the first studies to investigate this symbiotic relationship and for that credit is due.

Corbari, L., Durand, L., Cambon-Bonavita, M. A., Gaill, F., & Compère, P. (2012). New digestive symbiosis in the hydrothermal vent amphipoda Ventiella sulfuris. Comptes rendus biologies, 335(2), 142-154.

Heat-shock response of cyanobacteria

Cyanobacteria originated as a group of photoautotrophs nearly 3.5 billion years ago and were partially responsible for the initial oxygenation of the earth’s atmosphere. Today they are ubiquitous across aquatic ecosystems, even in extreme environments such as, hot springs, frozen lakes, salt ponds and deserts. They have the ability to survive in temperatures ranging from -60 to 74˚C. Studies on the stress responses of cyanobacteria are essential, as they play a major role in ecosystems as well as being used for human purposes in biotechnology and as biofertilizers.

Cyanobacteria are nitrogen fixers in which this process depends entirely on photosynthesis and both these processes are affected by heat and other stressors. Photosystems can become inactive as a result of a temperature shift, which has been shown in some unicellular cyanobacteria. Nitrogen fixation has also been found to be fragile at temperatures above 42˚C. These changes in metabolic capabilities in the presence of environmental stressors, emphasizes the importance of studying the heat shock response in cyanobacteria.

Cyanobacteria induce a group of proteins called heat-shock proteins (Hsps) by transcriptional activation and the degree of temperature increase determines the magnitude in which genes are upregulated. A typical heat-shock response in Synechocystis PCC6803 includes the upregulation of 90 genes after one hour of heat stress. The protein family Hsp100 are enhanced mainly by heat stress, whereas the Hsp90 family is associated with several abiotic stressors, a more general stress protein.

Molecular chaperones are essential components of many cellular functions: 1) folding of proteins after translation 2) transport of unfolded proteins 3) protein conformational homeostasis and 4) protection of the photosynthetic apparatus from stress induced damage. Hsps are present in low abundances under normal conditions, but are amplified when needed. These genes are then repressed by cis-elements or by trans-acting proteins. Maintenance of chaperone proteins in appropriate levels ensures there availability on demand and reduces any complications that might arise.

In conclusion, the roles of Hsp in cyanobacteria may help to enhance their stress tolerance, especially when subjected to sub-lethal levels prior to exposure. This was a fairly complex paper and went into a lot of detail about complicated pathways involving genes. The language used was also difficult to understand and maybe aimed mainly for geneticists.

Rajaram, H., Chaurasia, A.K. and Apte, S.K. (2014) Cyanobacterial heat-shock response: role and regulation of molecular chaperones. Microbiology. 160: 647-658

Gaping Jaw Disease - Are microbes to blame?

The Atlantic Hallibut (Hippoglossus hippoglossus) is an important commercial species, and is farmed in globally.  Although it has been commercially farmed for almost 40 years there are still issues arising in early developmet caused by opportunistic bacteria including Flexibacter ovolyticus, Vibrio salmonicida and Vibrio anguilarum.  One particular disease, known as “gaping jaw” is thought to be caused by bacteria infecting lesions caused by the rearing container in larvae.  s the larvae have very little immune system they are unable to fight it off and the disease itself eventually prevents effective feeding causing the animal to die from starvation. 

This paper looks at the analysis of the culturable bacteria found in larvae yolk sacs of those individuals exhibiting symptoms of gaping jaw.  Samples were taken from 40 larvae, grown on agar plates and amplified using PCR.  Although the results showed a heterogeneity for many bacteria, (32 out of 44 found), there were a number that were only found in larvae with gaping jaw.  These included species from Vibrio, Photobacterium, Flavobacterium and Bacillus genera suggesting that they may be linked to the jaw deformity.

The team also assessed the level of expression of three immune genes and found that expression was significantly elevated for two out of the three genes in larvae with gaping jaw.  One in particular, Hepcidin, was elevated 1196 times when compared to levels from healthy larvae.

One of the Vibrio species isolated had a high similarity to Vibrio alginolyticus which is a known pathogen.  In addition there are also previously described pathogens from the Photobacterium genera.  Even though Flavobacterium are generally harmless there are again a number that are associated with fish disease.  The presence of Bacillus however could be linked to the disease but there is also a possibility that they are there as an antimicrobial agent as they are able to produce bacteriocins.

The increase in levels of Hepcidin in the presence of the bacteria found on diseased larvae is interesting as it is know to up-regulate in other fish such as the Atlantic cod in response to bacterial infections.  Similar increases in levels of Hepcidin have also been seen in sea bass in response to infection from both Photobacterium and Vibrio species.

Given that previous studies into this disease have found little correlation between other environmental factors such as temperature, salinity and larval density it is possible that it is indeed caused by a bacterial infection.  Further study will now be required to determine which if any of these culturable bacteria may be responsible for the disease.  It would also be of interest to conduct metagenomic studies to determine which if any VBNC bacteria may be present and also complete individual tests to see what effect Hepcidin might have on the bacteria specific to the diseased larvae.

Urtubia, R., Gallardo, P., Lavin, P., Brown, N., & González, M. (2014). Characterization of culturable bacterial flora in yolk-sac larvae of Atlantic halibut (Hippoglossus hippoglossus L.) with" gaping jaws" syndrome. Latin American Journal of Aquatic Research, 42(1).

Copepods act as vectors for the for the accumulation of domoic acid in the Arctic food web

Out of the 37 described species of the diatom genus Pseudo-nitzschia, 12 are known to produce the neurotoxin domoic acid (DA), which accumulates in organisms which graze on the toxic Pseudo-nitzschia, such as copepods, krill, blue mussels and scallops, and are then passed through food webs when animals at higher tropic levels consume these grazers, and these even include whales and seabirds. Symptoms have even been found in humans that include several types of neurological disorders, and may even lead to mortality. One of these domoic-acid producing species, Pseudo-nitzschia seriata, was first discovered in Arctic waters in western Greenland. However, some of the earlier records of this species could have also included the morphologically-similar Pseudo-nitzschia delicatissima, which does not produce the toxin. P. seriata is a component of the Arctic phytoplankton community, but there is a lack of information about the frequency and intensity of blooms that form within Arctic waters.

Three species of copepod, Calanus finmarchicus, Calanus glacialis and Calanus hyperboreus are known to be the most important metazoan grazers in Arctic Ocean and are key sources of prey to shrimps, including the commercially significant northern shrimp (Pandalus borealis) which is consumed by a large proportion of the local human population of Greenland and other countries which import them. It also has recently been shown that there is a risk of domoic acid accumulation in higher trophic levels via Calanus spp., as the North Atlantic right whale (Eubalaena glacialis) was found to be regularly exposed to domoic acid from its critical prey source, C. finmarchicus, and this has been suggested as one of the possible reasons for the reduction in reproduction success of this critically endangered whale population.

The authors of this current study looked at the risk of transferring DA through the Arctic food web via the three different species of zooplankton to determine whether they accumulate the toxin when fed with unialgal toxic cultures. They also examined whether the copepods are affected by DA and if this can be measured by looking for changes in grazing rates and/ or mortality, whether the three copepods vary in grazing rate and toxin accumulation, and finally if the copepods ingest toxic and non-toxic Pseudo-nitzschia at the same rate.

They found that the three species of Calanus graze on and ingest toxin-producing Pseudo-nitzschia seriata and that they can accumulate domoic acid after feeding, so they are indeed possible vectors. This paper is also the first to study the consequences of grazing on Pseudo-nitzschia spp. in Arctic waters, as previous studies have not attempted to find differences in clearance or ingestion rates between the toxic and non-toxic species. 

The weight-specific carbon ingestion rates of all three Calanus species were not significantly different when they fed on either toxic or non-toxic species, but the toxic P. seriata was egested/ cleared out of the gut and also ingested significantly less at 6-12 h than at 0-6 h by C. finmarchicus and C. hyperboreus. The amount of specific fecal pellet production (SPP) were very similar following each type of diet, which confirmed that both the toxic and non-toxic species were grazed by the copepods in roughly the same quantities. Although the copepods in this study were fed with unialgal cultures, the authors say one of the most critical ideas to investigate in future research is determining whether copepods graze on toxic Pseudo-nitzschia and accumulate DA when given a choice of food in the natural phytoplankton community. In addition, C. finmarchicus and C. hyperboreus seemed to stop grazing during the last 6 h of the experiment, which could be due to physiological incapacitation caused by toxin ingestion.

There may be an effect of size of the host on grazing rate, as C. finmarchicus was the smallest of the three species, which may be why it had the highest amount of DA per gram per copepod, but it also had the greatest weight-specific carbon ingestion of the toxic species. However, the different size ranges of the diatom species were not predicted to influence the grazing rate of any of the copepod species. Not all DA is egested by gut clearance but part of it is actually assimilated and accumulated into the vector’s tissues, so even if the guts of the copepods are clear of all toxic Pseudo-nitzschia species, it may still get transferred up into the higher trophic levels when these zooplankton are consumed by predators.

This study demonstrated a new and interesting insight into the effect of toxic microorganisms on the lower levels of the food web in Arctic waters. They mention that there is a threshold level where the Calanus species start to be affected by the toxin themselves, which seems to depend on a number of factors, including the size of copepod and concentration of domoic acid. In addition, the frequency and intensity of Pseudo-nitzschia species within Arctic waters is unknown. So I wonder whether certain external/ environmental conditions can enhance this impact, for instance in the face of global warming. An increase in sea temperature could cause a possible rise in phytoplankton blooms, and this could either cause a higher amount of DA to accumulate in copepods, or could lead to greater incidences of mortality. Either of these options could potentially impact the food web in some way, so the effect of temperature on the growth of blooms, and the subsequent influence of domoic acid on Calanus species and higher trophic levels, should be investigated.

Tammilehto, A., Nielson, T.G., Krock, B., Møller, E.F., and Lundholm, N. (2012) Calanus spp. – Vectors for the biotoxin, domoic acid, in the Arctic marine ecosystem? Harmful Algae, 20: 165-174