"Jamming the radar"

Over the last two decades aquaculture has become the fastest growth sector of agribusiness worldwide. However, aquafarming is facing one fundamental problem: Diseases. The aquaculture industry loses billions each year because of diseases caused mainly by microbial infection. In order to counter this dilemma the use of antibiotics has been widespread. Their excessive use has generated unwanted side effects. Therefore, an alternative solution is necessary. One potential solution could be quorum quenching (QQ). Many species of bacteria use quorum sensing (QS) to regulate their gene expression in response to cell-population density. Behaviors such as virulence controlled by QS only occur at high bacterial cell densities. Bacteria use autoinducers like N-acyl-homoserine lactone (AHL) for intraspecific communication. QQ enzymes which interfere with the QS system by degrading signal molecules can shut down the expression of pathogenic genes.

Recent studies have shown that there are quite a few marine bacteria with the enzymatic potential of QQ. Zheng et al. (2016) conducted a study with focus on the Pacific white shrimp (Litopenaeus vannamei). The main focus of this study was to compare the bacterial composition of different health statuses. Further, they looked at the bacterial composition of distinct larval stages. 240 bacterial strains where identified in total with a significant shift between samples from healthy and diseased populations and also within different growth stages.

What I found most interesting was that they screened the 143 strains which they isolated from healthy shrimp and water samples for their AHL degrading activity. 18 out of 143 strains showed reduction of the normalized β-galactosidase activity. The AHL level is proportional to β-galactosidase activity. Ten out of 18 strains showed strong AHL degrading potential. The dominant species with strong degrading potential were Tenacibaculum mesophilum (nine strains) and Microbacterium aquimaris (two strains). They suggest that the identified bacteria with strong degrading potential could potentially be test to control diseases in aquaculture.

In my opinion these findings are very promising. Previous studies have revealed that the use of organisms with the potential of QQ can be an effective way to block QS systems of bacteria. E.g. QQ can be used to control biofilm formation and therefore prevent biofouling (Joint et al. 2007). Additionally, the author mentioned recent studies where QQ was successfully applied to marine organisms such as the giant freshwater prawn. Anyway, further research is necessary. More bacteria with QQ potential for other farmed species have to be identified. Further, the efficiency in commercial aquaculture has yet to be proven.

If you are interested in that topic: I found two useful reviews about QQ enzymes and QQ marine agents:

Tang et al. (2014), Quorum Quenching Agents: Resources for Antivirulence Therapy, http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4071575/

Chen et al. (2013), Quorum Quenching Enzymes and Their Application in Degrading Signal Molecules to Block Quorum Sensing-Dependent Infection, http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3794736/

And here’s the reference for this article:

Zheng Y., Yu M., Liu Y., Su Y., Xu T., Yu M., Zhang X. (2016) Comparison of cultivable bacterial communities associated with Pacific white shrimp (Litopenaeus vannamei) larvae at different health statuses and growth stages, Aquaculture,doi:10.1016/j.aquaculture.2015.09.020

http://www.sciencedirect.com/science/article/pii/S0044848615301794

Phytoplankton and neurotoxin. More complicated than it sounds.

Recent studies show that dinoflagellates have acquired a number of important things from cyanobacterial horizontal gene transfer, for example RuBisCo; a crucial component in CO₂ fixation, histone-like proteins and other plasmid-related genes. As certain dinoflagellates are also known to produce harmful compounds known as saxitoxins, Hackett et al. carried out genomic analysis of the dinoflagellate Alexandrium tamarense to look for saxitoxin genes (sxt), testing the hypothesis that they too had been passed on by horizontal gene transfer.

The researchers first grew A. tamarense in media and harvested RNA which was prepared and sequenced. The genetic material was then screened for a particular domain found in cyanobacterial stx genes.

The study showed that A. tamarense had 13 homologous genes for cyanobacterial saxitoxin production, however the genes were not structurally similar. In cyanobacteria, sxtA is composed of two domains fused together, in the dinoflagellate however, the two domains are separate proteins, suggesting the two are not related phylogenetically. Other proteins that showed similarities to cyanobacterial proteins were not calculated to have arisen by horizontal gene transfer to the dinoflagellate. 

These results tie in nicely with another article Laura blogged about recently. Her article said that the dinoflagellate Gymnodium catenatum did not produce saxitoxin without its bacterial community. G. catenatum is mentioned briefly in the discussion of this article as having sxt homologues. So how do we interpret these results? Without being too speculative, I think it may be possible that the dinoflagellate doesn't have all the proteins required to produce saxitoxin and therefore requires its bacterial community to create it instead. 

Another idea put forward in this paper is that the dinoflagellate enzymes have evolved independently of the cyanobacteria and are functionally different despite structural similarities, that is, the dinoflagellate sxts are involved in other processes separate from production of saxitoxin. A next step would be to test the functionality of the genes from these dinoflagellates to see if they do or don’t produce the toxins. 

Here’s a link to Laura’s blog, if you want to read it yourself:

http://2015-mbio322.blogspot.co.uk/2015/11/algae-and-bacteria-toxic-relationship.html

And here’s the reference for this article: 

Hackett, J.D. Wisecaver, J.H. Brosnahan, M.L. Kulis, D.M. Anderson, D.M. Bhattacharya, D. Plumley, F.G. and Erdner, D. (2013) Evolution of Saxitoxin Synthesis in Cyanobacteria and Dinoflagellates. Molecular Biology and Evolution 30(1) pp.70-78.

If you have any questions or comments, do write them below and I’ll respond as best I can.

Phage Therapy: hero or zero?!

 Scleractinian  corals are host to a diverse microbial consortia that has been found to play a number of roles from mutualistic symbionts to vectors of disease. In fact, the coral microbiota is thought to be a major part of the holobiont and so has repeatedly been posited to play a pivotal role in coral response to changing global environments.

 Unfortunately, the diversity of the coral microbiome also comes hand in hand with its complexity and therefore, in spite of the fact that there have been over three dozen coral diseases described, the aetiological agent behind the majority is still unkown. This phenomenon has largely been attributed to the ‘compromised host’ hypothesis which suggests that many coral diseases are causes by an accumulation of factors that lead to stress-induced vulnerability to virulence factors. In order to understand the mechanisms driving this, the bacterial composition of the holobiont has been studied across all coral health states. However, what has yet to be examined is the bacteriophage community associated with corals, along with its effect on coral health.

Therefore, in a study by Soffer et al, the coral associated bacteria and bacteriophages are simultaneously examined in order to further understand their role in coral disease. But why look at phages? Well, in addition to the fact that lytic phages are thought to cause around 10²⁸ infections, some lysogenic phages have been implicated to play a beneficial role in fighting off infection to coral disease. Because of this many have put forward the idea of ‘phage therapy’ as a means for treatment of coral diseases. And, although it makes sense to use phages to target disease-casing microbes, the interactions involved in this top-down means of control are largely underexplored. The study therefore uses a combination of 16S rRNA gene amplicon and shotgun metagenomics analyses to investigate in situ variances in phage and bacterial populations across coral health states, in order to identify possible candidates for use in phage therapy.

The study identified six phage strains that exclusively targeted two bacteria which are enriched in white plague disease which implicates them as potential candidates for phage therapy. In addition to this, it was discovered that coral mucus contains receptors for bacteriophages and therefore may retain beneficial phages, a condition which may be interrupted in a diseased state. All of this indicates potential for the use of phage-therapy as a means of controlling disease-associated bacteria in cases where host immunity has been compromised.

It is my opinion, however, that the use of phage therapy as an in situ means for disease control in environmental corals is not possible at this time. This is due in part to the fact that many of the target hosts simply aren’t amenable to culturing, a fact which means the correct phages may be difficult to identify. When coupling this with varying levels of phage specificity and bacterial host susceptibility it is clear that phage therapy may not be ready for practical applications. This paper does however highlight its potential, as well as helping to gain further insight into the mechanisms behind coral immune response.

Reference:

Soffer, N, Zaneveld, J. and Thurber, R. V. (2015). Phage-bacteria network analysis and its implication for the understanding of coral disease. Environmental Microbiology. 17 (4), 1203-1218.

Civil War

Apostichopus japonicus is a commercially important species of sea cucumber found commonly in the shallow waters of south east Asia. In countries such as Japan and China, it is a popular food source and is both fished and farmed, having created a large aquaculture industry based around it. As is common in all types of farming and aquaculture industries, A. japonicas has had its fair share of disease outbreaks, thought to be aggravated in these regions by a substantial increase in the farming industry, which is increasing output and pollution.

Vibrio splendidus is a common pathogen of A. japonicas, causing skin ulceration syndrome, which is a major limitation in the farming of this species. Previous studies have looked into using various probiotics as a means of efficiently treating V. splendidus infections, and while they have proven effective at reducing the growth of V. splendidus, all of these tests have been carried out in vitro, and the efficiency at which they would perform in vivo is largely unknown.

(F. Yan, 2014) presents the results of the probiotic bacteria Paracoccus marcusii DB11 when used as a dietary supplement on the health of A. japonicus juveniles. The team exposed A. japonicus to DB11 via their feed at varying doses, and measured the levels and activities of 5 immune enzymes, the sea cucumbers growth rates, and the survival rate when infected with V. splendidus. They discovered that even the lowest dose of DB11 had positive effects on all factors measured, and growth rate increased with increasing doses of DB11, as did all immune enzyme activity. Additionally, at the highest dose of DB11 administered, there was 0% mortality when the juveniles were infected with V. splendidus, and only 15% mortality at the lowest dose, which could either suggest inhibition of the growth of V. splendidus, or inhibition of pathogenicity. These results are very promising when compared to a 40% mortality rate when the juveniles were left untreated, which undoubtedly has huge impacts on the aquaculture and farming of these organisms.

Using probiotics in aquaculture is a relatively new but very promising field, and while studies still seem to be in their infant stages (with most experiments being carried out in vitro), I think this paper provides a very useful insight into a real environmental use, which is potentially much less harmful and costly than the use of antibiotics and other previously tested methods to improve the health of organisms from aquaculture. I believe this paper gives some good indicators as to what to look for and how to conduct and efficiently apply these studies in the future, and could present an economically viable solution to many disease problems faced in aquaculture.

F. Yan, X. T. (2014). Growth performance, immune response, and disease resistance against Vibrio splendidus infection in juvenile sea cucumber Apostichopus japonicus fed a supplementary diet of the potential probiotic Paracoccus marcusii DB11. Aquaculture, 105-111.

Combating Vibrio harveyi infections in shrimps with microalgae supplements

Disease outbreaks (particularly Vibrio species) in aquaculture can affect production and trade in various parts of the world. There has been much development into antibiotic resistance, however the popular use of antimicrobials for disease control has been seen to have hazardous effects to human health, as it encourages bacterial resistance. Studies have shown that the use of microalgae as live feed in aquaculture can reduced the levels of bacterial diseases. This study by Seraspe et al., (2014) looks at using Chaetoceros calcitrans, a unicellular and eukaryotic diatom, as a supplement in the diet of Penaeus monodon to control Vibrio harveyi infection. Total lipid and fatty acid profile of Chaetoceros calcitrans was also analyzed to determine the presence of polyunsaturated fatty acids (PUFAs) which are considered important in the treatment and prevention of diseases. 

Cultures of Chaetoceros calcitrans were obtained, freeze-dried and mixed to form the basal diet at 15 and 30g/kg diet. Feed incorporated with Curdlan was used as a positive control. Penaeus monodon postlarvae were obtained and fed all the same feeds up until an average weight of 10g, and then were fed either Chaetoceros calcitrans (15g/kg diet or 30g/kg diet) or a control of Curdlan. They were then subjected to a bacterial challenge test with virulent Vibrio harveyi (via intramuscular injection). Relative percentage survival (RPS) was computed based on a formula. Immune responses were noted after exposure including prophenoloxidase activity (proPO) which involved the production of protein and as an index to measure general activation of the immune response, along with plasma protein concentration. 

Results showed that the microalgal diet did not affect general growth and survival of the shrimp. However, after exposure to the Vibrio harveyi challenge, the microalgal supplementation did affect the shrimp’s survival. Decreased mortality rates were seen in shrimps fed the supplement of Chaetoceros calcitrans, along increased span of time till 80% mortality was reached (if occurred). There was also higher prophenoloxidase activity in shrimps fed Chaetoceros calcitrans, indicated an enhanced immune response against bacterial diseases. This could be due to the high fatty acid content found in Chaetoceros calcitrans, which are known to have antibacterial effects.

Overall, the use of microalgae as a supplement in preventing and treating bacterial diseases could provide opportunities against infectious diseases, particularly with Vibrio species in aquaculture. This could possibly be incorporated into marine habitats themselves, especially with how the increase in sea-surface temperature comes with an increase in Vibrio species.

Reference:

Seraspe, E., Gabotero, S., De la Peña, M., Pahila, I. and Amar, E. (2014). Evaluation of dietary freeze-dried Chaetoceros calcitrans supplementation to control Vibrio harveyi infection on Penaeus monodon juvenile. Aquaculture, 432, pp.212-216.