Another One Bites the Dust

Terrestrial dust provides biologically important nutrients to the marine environment and facilitates the transportation of microorganisms. In the aftermath of a huge dust storm on the east coast of Australia in 2009, masses of fungal spores and mycelia formed a “floating raft” that covered an area stretching across coastal waters from Sydney to Brisbane, covering an area equivalent to 25 times that of the UK. Using molecular sequencing of three different genes these spores and mycelia were identified as Aspergillus sydowii. The pathogenic strains of A. sydowii have been associated with huge sea fan mortality in the Caribbean and are known for having an adverse effect on Symbiodinium dinoflagellate coral endosymbiont motility.  

Molecular genetic analyses have shown no clear difference between pathogenic and non-pathogenic strains of A. sydowii. Hayashi et al (2016) therefore set out to examine the metabolic profile of isolates from the 2009 Australian dust storm aftermath and compare them to the metabolic profiles of those from terrestrial habitats. They also wanted to explore the fungal diversity on the 2009 “raft” and further look at how A. sydowii metabolites affected various strains of Symbiodinium, in terms of photosynthetic performance by measuring the maximum quantum yield (Fv/Fm).

The metabolites were isolated using High Performance Liquid Chromatography (HPLC) and analysed against a library of known metabolites and a type species library. Strains of the Symbiodinium dinoflagellate were obtained from the Australian National Algal Culture Collection (ANACC) and selected based on genetic clade, growth rate and geographic region (Clade A – Heron Island GBR Australia, Clade C – Hawaii US, Clade A1 – Palau). Four typical A. sydowii metabolites (sydowinol, sydowinin A, sydowinin B and sydowic acid), as well as crude extracts of A. sydowii were added to individual microplates and cultured cells of Symbiodinium were added. The maximum quantum yield was measured after every second day over 8 days.

Results showed that, of the raft species, A. sydowii was the most dominant but other species of Aspergillus along with species of Penicillium and Cladiosporium accumulated as secondary colonisers. HPLC analyses showed that >50% major metabolites were shared between terrestrial and marine strains of A. sydowii but the marine had a more streamlined metabolic pathway, which suggested intensive strain selection on marine adaptation. Also, all metabolites reduced Fv/Fm of Symbiodinium dinoflagellates with sydowinol and sydowic acid the most active in doing so. Crude extracts exhibited less clear effects on Symbiodimium Fv/Fm. Finally Symbiodinium Clades C and A exhibited high and moderate sensitivities, respectively, with Clade A1 showing low sensitivity to metabolites.

From this study the authors suggested that the Great Barrier Reef has not experienced significant coral disease events compared to those experienced in the Caribbean due to the higher octocoral diversity found there. Of the 8 clades of Symbiodinum, this study only looked at 3 and so although this study provides quite a good basis for further investigation into this area, some of the conclusions drawn cannot made based on the data collected in this study. Further studies in Symbiodinium clades could yield better data for a more direct comparison with Caribbean corals but in general this paper is a good foundation for future study.

Paper reviewed

Hayashi, A., Crombie, A., Lacey, E., Richardson, A.J., Vuong, D., Piggot, A.M., Hallegraeff, G. (2016). Aspergillus Sydowii Marine Fungal Bloom in Australian Coastal Waters, Its Metabolites and Potential Impact on Symbiodinium Dinoflagellates. Marine Drugs 14 (3) 59

Viruses and Ctenophores

Gelatinous zooplankton plays an important role in marine and brackish ecosystems. Cnidaria and Ctenophores are not only predators and invaders, but their blooms also affect human infrastructure, tourism and aquaculture negatively. Over the last half century an increase in gelatinous zooplankton has been recorded in most marine ecosystems, however, their population regulation mechanisms are still relatively unknown. Ctenophores harbour specific bacterial communities as well as invertebrate parasites and symbionts, yet the presence of viruses had not been previously reported. In their paper, Breitbart et al. (2015) analysed the presence of circular, Rep-encoding single-stranded DNA (CRESS-DNA) viruses in two ctenophore species. 

Specimens of Mnemiopsis leidyi and its predator Beroe ovata were collected from February to October 2013 in an estuary in coastal Georgia, USA. The ctenophores were dissected and the extracted DNA was amplified with rolling circle amplification (RCA). Subsequently, the DNA was digested with restriction enzymes, cloned and Sanger sequenced. The prevalence of viral genomes over the 8-month period was examined using PCR assays. 

Out of the 17 extracted circular DNA molecules (between 1030-2838 NT), nine were not definitively identifiable and were not further analysed. The identified CRESS-DNA molecules were characterized by a Rep-encoding ORF and a supposed ori with a conserved motif. After discarding potential partial genomes and plasmids, Breitbart et al. (2015) identified four ctenophore-associated circular viruses (CtaCV-1 to CtaCV-4). These CRESS-DNA genomes were similar to viral sequences identified in previous metagenomic analyses.

Interestingly, while the CtaCVs were retrieved from the same species, they were as distantly related to each other as they were to CtaCVs in other marine organisms and ecosystems. These results reflect prior observations, that CRESS-DNA viruses seem to show no apparent clusters of location, environment or host organism. 

The analysis of CtaCVs over an 8-month period showed temporal shifts of dominant viral species in M. leidyi. While some viruses (CtaCV-2 and CtaCV-3) were only detected during certain months, others (CtaCV-1 and CtaCV-4) were present over the whole of 8-months. However, ctenophore length and volume did not seem to affect viral presence. Analysis of B. ovata associated viruses showed a smaller viral community without CtaCV-3. To exclude the possibility of copepod prey introducing viruses into their predators, copepod samples were screened for CtaCVs. Moreover, the stomachs of the ctenophores were removed during dissection and not further analysed. The results showed a much lower abundance of CtaCVs in copepod batches compared to individual ctenophores, which would make it unlikely for prey to falsify the results. 

Since the increase of gelatinous zooplankton has many negative effects, more information on its population dynamics is sorely needed. This study aims to address one of the gaps in our knowledge. Moreover, the sequence data provided enables quantitative studies of ctenophore associated viruses and their potential roles in regulating ctenophore populations. It would be interesting to see if follow-up papers might be able to shed more light on this subject. 

However, this paper also has its limitations, namely small sample size and no variation in location. Furthermore, I would be hesitant to discount the possibility that the viruses were introduced via copepod prey, as the viruses could have transferred from the stomach to the comb rows of the ctenophores. I would have liked to have seen an analysis of the CtaCV abundance in the stomach compared to the comb rows.

Reviewed Paper:

Breitbart, M., Benner, B. E., Jernigan, P. E., Rosario, K., Birsa, L. M., Harbeitner, R. C., ... & Berger, S. A. (2015). Discovery, prevalence, and persistence of novel circular single-stranded DNA viruses in the ctenophores Mnemiopsis leidyi and Beroe ovata. Frontiers in microbiology, 6. Link: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4683175/

Further Reading:

Tijssen, P., Pénzes, J. J., Yu, Q., Pham, H. T., & Bergoin, M. (2016). Diversity of small, single-stranded DNA viruses of invertebrates and their chaotic evolutionary past. Journal of Invertebrate Pathology, 140, 83-96. Link: http://www.sciencedirect.com/science/article/pii/S0022201116301318

Make reefs great again: Symbiodinium clade D trumps Vibrio

Coral are inhabited by a diverse consortium of microbial life, including Symbiodinium, a genus of symbiotic dinoflagellate. There are nine identified clades of Symbiodinium (A-I), some of which can be found in coral tissues as mono or multi-clade populations. Corals can associate with different clades, and many studies have looked at the effect of different clades on the ability of corals to deal with stressful environments and disease. Over the last 30 years the frequency of coral disease outbreaks and bleaching events has increased, so understanding how different members of the coral holobiont affect the coral’s resilience to disease or ability to acclimate is increasingly important.

Rouzé et al investigated the Symbiodinium assemblage in the Indo-pacific stony coral Acropora cytherea and measured how this affected the corals sensitivity to disease (broadly described as ‘white disease’ but comprising any disease causing chronic lesions) and whether the presence of disease was linked with the appearance of Vibrio spp. Several small fragments were taken from eleven colonies of A. cytherea every 2 months for just over a year, and total coral DNA was extracted (this excludes DNA from outside coral tissues i.e. in the mucus). Symbiodinium clades were detected using clade-specific primer sets for clades A-F, and presence of Vibrio spp. were detected using genus specific 16S rDNA primers. Clades A, D and C were found in the tissues of A. cytherea, but B and F were absent despite being present in the surrounding environment.

Roughly half of the colonies (five out of eleven) developed disease lesions consistent with white syndrome during the study, and four of those five colonies contained only clade A when symptoms were identified. The colonies that remained healthy throughout the study contained A and D simultaneously, except for one which also contained clade C.

Vibrio spp. were only detected in diseased corals, but no link between these results can be made because Vibrio spp. were detected before, during and/or after disease symptoms, suggesting that they are not the causative agent of the disease. This does however strongly suggest that the bacteria may be opportunistic. During Vibrio spp. infection, there was a switch from multi-clade association to mono-clade A, and vice versa if the bacteria disappeared. The results show that affected colonies of A. cytherea were either lacking clade D to begin with, or lost it over the duration of the study. The strong negative correlation between association with clade D and Vibrio spp. infection may reinforce the hypothesis that the clade has antibacterial properties.

Although this study has significant results showing that corals associated with mono-clade A are more susceptible to white disease and Vibrio spp. infection, the small sample sizes give the results less weight. From only four colonies containing mono-clade A, the authors suggest that this makes the coral sensitive to white disease. Infection mainly occurred during the hottest months, leading me to believe that the ‘detrimental, opportunistic clade A’ may only become a hindrance when the environment becomes very stressful. If clade A is indeed a detrimental member of the microbiome it is strange that it occurs most frequently out of all the clades in this species of coral. This study is however very in-depth and with more replicates, as well as studies on different species of coral to see if the benefits and drawbacks of different clades are consistent, it could help us to understand how corals react to stress and disease.

Rouzé H., Lecellier G., Saulnier D., Berteaux-Lecellier V. (2016) Symbiodinium clades A and D differentially predispose Acropora cytherea to disease and Vibrio spp. colonization. Ecology and Evolution. 6(2) 560-572

Dodgy tummy from eating oysters? Curcumin is the answer!

Vibrio parahaemolyticus is a gram negative bacteria which can be found in brackish and marine environments, most notably along coastlines when the water temperatures reach their peak during the summer months. It has been known to infect humans via a range of different pathways including through open cuts and wounds, and through areas such as the eyes and ears, and has even been linked to a few deaths in the Hurricane Katrina disaster. However, the most common problem associated with this bacteria is the cause of gastroenteritis in humans when undercooked or raw oysters are ingested. There have been many documented outbreaks of this, and when looking at the evergrowing problem of climate change, the outbreaks appear set to become more frequent. Thoughts of reducing levels of Vibrio parahaemolyticus before consumption of raw oysters have been in circulation for some time, and usually involve the use of non-thermal methods to decontaminate the pathogen.

Wu et al (2016) also thought about this, and set out to reduce the levels of Vibrio parahaemolyticus using antimicrobial photodynamic therapy (aPDT) – a photochemical reaction involving the interaction of a photosensitizer (PS), visible light and oxygen, as this has been shown to destroy bacterial cells of other gram negative bacteria. Curcumin was also added as it has been shown to become activated by visible light and inhibit the growth of bacterial cells. Thus, the objective was to find out if curcumin-mediated photodynamic action would inactivate Vibrio parahaemolyticus cells. The experiment was divided into four groups: negative control (no PS, no light irradiation, L-S-), LED irradiation (no PS, same irradiation as aPDT samples, L+S-), curcumin treatment (10mM, PS, no irradiation, L-S+) and LED irradiation plus curcumin treatment (5mM, 10mM, 20mM, L+S+).

The results showed that curcumin-mediated photodynamic action significantly killed Vibrio parahaemolyticus cells. The viability of Vibrio parahaemolyticus significantly reduced in L+S+ group compared to L-S- group, with a lethal rate of 100% at the 10mM and 20mM concentrations. The viability was also significantly reduced in L+S+ group compared with L-S+ group. This showed that curcumin-mediated photodynamic action worked faster and was better at inactivating the bacterial cells with both LED irradiation plus curcumin treatment, especially at the higher curcumin concentrations.

I think this paper has opened a gateway for other scientists to continue this work using this method to inactivate Vibrio parahaemolyticus cells, and future work to inactivate the bacteria whilst inside the oysters may even show the number of human cases infected with the bacteria from these oysters to significantly decrease. The method they used on this particular pathogen hadn’t been used before this study, and yet it proved to be successful in giving the results the scientists were hoping for. Due to this, I expect to see this method utilized in other similar studies in the near future.

Reviewed paper: Wu, Juan., Mou, Haijin., Xue, Changhu., Leung, Albert Wingnang., Xu, Chaunshan., and Tang, Qing-Juan. (2016). Photodynamic effect of curcumin on Vibrio parahaemolyticus. Photodiagnosis and Photodynamic Therapy. 15: 34-39.  http://www.sciencedirect.com/science/article/pii/S1572100016300539

Chinese Whispovirus: A chitin-binding protein is involved in White Spot Syndrome

White Spot Syndrome (WSS) is a viral disease of crustaceans. Outbreaks of WSS can be devastating to aquaculture yields and severe infections in Asian penaeid prawn farms have pushed facilities to near collapse. The etiological agent responsible for WSS is an enveloped, lytic dsDNA virus known as Whispovirus (WSSV) which causes tissue necrosis culminating in host mortality. There exists, therefore, a heavy economic incentive to investigate the pathology of WSSV and intensive research has been conducted to understand the interaction of WSSV and its host to design an effective treatment. Recent work by Li et al, (2016) has identified a chitin-binding role in a major envelope protein that the authors believe is integral to infection.

The cramped, squalid conditions of shrimp aquaculture places healthy shrimp in close proximity to the carcasses of the infected and deceased. Due to the cannibalistic nature of penaeid prawns, the authors hypothesised that ingestion may the primary route of viral infection. Large areas of the prawns’ digestive track are coated with a chitinous lining, and chitin-binding by WSSV may facilitate infection, however little is known about chitin-binding and host invasion in this virus. Therefore, the authors investigated the ability of four major envelope proteins to bind to chitin.

Recombinant proteins were expressed in transformed E. coli and incubated with chitin beads. Subsequent immunoblotting revealed that one of the proteins, VP24, exhibited chitin-binding and became a prime candidate for further investigation. Following a mutagenesis screen, the chitin-binding site of VP24 was identified as a 14 amino acid sequence and the authors explored the possibility of exploiting this region to aid in the treatment of WSS. A synthesised peptide of the same sequence blocked the ability of recombinant VP24 to bind to chitin when co-incubated with chitin beads and a subsequent in vivo experiment strikingly showed that oral inoculation of the host with the binding peptide significantly decreased the infectivity and viral load of WSSV in Whiteleg Shrimp (Litopenaeus vannamei). Such peptide inoculations could prove vital in treating WSS across the globe. 

Overall, this study employed a diverse methodology to achieve an exciting insight into the molecular interaction of the WSSV ‘infectome’. Their discovery of a chitin-binding role in VP24 and synthesis of a chitin-binding therapeutic peptide is tantalizing and, while preliminary, could play a major role in our understanding and treatment of WSS. Chitin-binding is implicated in various marine diseases (such as in various vibrioses) and therefore understanding this phenomenon may provide insights into treating other infections. 

This is, however, not the first treatment for WSS proposed by researchers and previous attempts to treat the disease have ranged from DNA vaccinations (Ning et al, 2009) to medicinal herb extracts (Citarasu et al, 2006). A previous attempt to orally inoculate shrimp with WSSV envelope proteins too reported success as far back as 2004 (Witteveldt et al, 2004) but WSS is still devastating shrimp industries. While this treatment involves a different protein and administration technique, time will have to tell as to whether this discovery can make a significant contribution to protecting shrimp aquaculture or whether it is yet another false lead. Nevertheless, this paper makes a significant advancement in our understanding of a globally and economically important pathogen and offers another step closer to an effective treatment. 

Reviewed Paper: Li, Z., Li, F., Han, Y., Xu, L., & Yang, F. (2016). VP24 is a chitin-binding protein involved in white spot syndrome virus infection. Journal of virology, 90(2), 842-850. http://jvi.asm.org/content/90/2/842.short

DNA Vaccine: Ning, J. F., Zhu, W., Xu, J. P., Zheng, C. Y., & Meng, X. L. (2009). Oral delivery of DNA vaccine encoding VP28 against white spot syndrome virus in crayfish by attenuated Salmonella typhimurium. Vaccine, 27(7), 1127-1135. http://www.sciencedirect.com/science/article/pii/S0264410X08015715

Herbal Remedy: Citarasu, T., Sivaram, V., Immanuel, G., Rout, N., & Murugan, V. (2006). Influence of selected Indian immunostimulant herbs against white spot syndrome virus (WSSV) infection in black tiger shrimp, Penaeus monodon with reference to haematological, biochemical and immunological changes. Fish & shellfish immunology, 21(4), 372-384. http://www.sciencedirect.com/science/article/pii/S1050464806000088

Previous Oral Inoculation: Witteveldt, J., Cifuentes, C. C., Vlak, J. M., & van Hulten, M. C. (2004). Protection of Penaeus monodon against white spot syndrome virus by oral vaccination. Journal of virology, 78(4), 2057-2061. http://jvi.asm.org/content/78/4/2057.short s

Saving coral reefs with phage therapy from Vibrio coralliilyticus?

Coral reefs build large underwater ecosystems. In the last decades they have to struggle more and more with the consequences of global warming as the spread of coral disease often related with a pathogen of the Vibrio genus. One of these pathogens is Vibrio coralliilyticus which was isolated from different geographical regions and though its influence on corals is an important point to investigate. With respect to the fact that coral disease cannot be treated with antibiotics and that corals do not have an adaptive immune system phage therapy might be a reasonable way to prevent coral disease which was investigated by Cohen et al. (2013). 

In this study the V. coralliilyticus strain P1 was used, so was a purified bacteriophage termed YC isolated from infected corals. Both were found on infected corals and in the seawater around at the Great Barrier Reef, Australia. 

This study was split up in three main parts. The first one was the identification of the phage. The phage was isolated from seawater of the Great Barrier Reef and observed with TEM to determine its morphology. That shows that it belongs to the Myoviridae family. Purification of the phage nucleic acid and gel electrophoresis were used to show that the phage is a DNA phage with a small genome size of 11 kb. Furthermore Cohan et al. observed that the absorption rate amount 500 phage particles per bacteria cell per minute. The phage was termed bacteriophage YC. 

The second part was the investigation of the effect of V. coralliilyticus strain P1 on the corals’ endosymbiont Symbiodinium. The photosynthetic activity was measured by using an iPAM fluorometer. A decrease of the photosynthetic activity was observed in the treatments with P1. After 24 h Cohen et al. measured a reduction of 90 % in the photosynthetic activity. There was no effect detected when P1 was treated with Phage YC before adding to Symbiodinium. 

To examine the efficiency on living corals 6 month-old juvenile corals were observed. They were raised from larvae and then brought out in the reef water of the Great Barrier Reef. At age of 6 months they were recollected and positioned in aquaria. They activity was indirectly measured by investigating their photosynthetic activity using an iPAM fluorometer as before. Changes in morphology were observed by using a dissecting microscope and a digital camera. When the juvenile corals were treated with P1 they immediately retracted their polyps and after 1 h they began to expel their symbionts. After 9 h only the skeleton remained. The addition of Phage YC to P1 before adding to the juvenile corals changed the results. No changes were detected in the 12 h experimentation period. After 12 h a slight increase of photosystem inactivation and also expulsion of Symbiodinium and tissue lesions were observed. 

The study suggest that the addition of Phage YC leads to a lysis of V. coralliilyticus P1 what result in an inactivation of the Zn-metalloprotease of the bacteria that normally leads to an inhibition of the photosystems in Symbiodinium. All in all Cohen et al. conclude that Phage YC is a good treatment to prevent or stop spread of coral disease triggered by Vibrio coralliilyticus but that further research is needed to examine whether this phage treatment is feasible and possible in open coral reef systems. 

This paper can be an approach on preventing spread of coral disease. Having said this, it needs much more research on the long-time effect of phage therapy. It is interesting that phage treatment has a positive effect for a short-time period of 12 h as investigated in this study but slightly negative effects were shown after 12 h in the juvenile corals. This study does not seem complete because in their conclusion Cohen et al. do not mention these negative effects. This gives the impression that the researches have not thought on long-time effects and they wanted to publish as soon as possible without finishing their work. As a conclusion I would say that this study is a base for further studies on long-term effects of phage treatment where it can be shown that phage treatment might not be as effective as suggested by Cohen et al.

Reviewed paper:

Cohen, Y., Joseph Pollock, F., Rosenberg, E., & Bourne, D. G. (2013). Phage therapy treatment of the coral pathogen Vibrio coralliilyticus. Microbiologyopen, 2(1), 64-74.