Viruses Manipulate the Marine Environment

A review of: Rohwer, F., and Thurber, R. V. (2009) Viruses manipulate the marine environment. Nature. 459; 207-2012

This paper is a review of current understanding into the ways in which marine viruses can affect their hosts and their environments. The importance of these studies focuses on the influences of marine viruses on both biogeochemistry and host evolution which as economic and conservation related implications. Marine viral diversity is a major area of current study but is difficult to pursue due to viruses lacking universally conserved genes or being difficult to culture. However, techniques such as pulsed field gel electrophoresis and shotgun sequencing have been developed to overcome these problems and it has been found that viruses are astoundingly diverse with more than 5000 viral particles per 100 litres of seawater.

Viruses can carry and transfer host genes and are capable of modifying host physiology with both positive and negative consequences for the host. Horizontal gene transfer between the virus and the host is a consequence of viruses acting as gene reservoirs for the host. An example of this is in cyanobacteria in which bacteriophages contain genes for photosynthesis which they use to maintain photosynthesis in the cyanobacteria during viral infection. The phages use the energy generated from photosynthesis for viral production in the host.

Viruses can also manipulate their hosts and transform them from benign microorganisms to pathogens through the high number of virulence genes that viruses contain, including antibiotic resistance, toxicity and host invasion, found in metagenomic studies. Bacteria can extend their ecological niches by taking up these genes, such as Vibrio cholera, a usually harmless marine bacteria that becomes a pathogen which it incorporates the viral cholera toxin genes.

Studies have found that the rate of transduction between the viruses and their hosts is high with data suggesting that as many as 10²⁴ genes are moved this way each year in the world’s oceans. However, this is actually thought to be higher due to generalised transduction agents (GTAs), similar to bacteriophages but smaller in size and number of genes. They contain only the host DNA which they then inject into the recipient. This is effective transduction as it not only allows gene swapping between organisms but between ecosystems too. It is also thought to contribute to niche differentiation in closely related species.

Viruses are expert manipulators, of protists, metazoans and even themselves. Examples in this paper include Coccolithophores, a group of eukaryotic phytoplankton, with calcium carbonate scales known as coccoliths. The Coccolithophores have two life stages, one (diploid) involves the Coccolithophore in its coccolith shell and the second (haploid) is its sexual stage where the Coccolithophore is naked, can be motile and is resistant to the viruses that can infect in the diploid stage. Coccolithophores’ importance lies in their blooms which influence global temperature and remove atmospheric carbon dioxide when their coccoliths sink. This boom and bust system is primarily influenced by the infection and lysis of Coccolithophore specific viruses, whose genomes contain genes for an apotosis pathway that was once designed by the Coccolithophores to prevent the spread of viruses and but is now used by the viruses to facilitate it. Studies on these have led to formation of a hypothesis which suggests that the host and the virus will continually evolve to become resistant to the other in an 'arms race' until the Coccolithophore moves into its haploid stage and evades the virus altogether.

The solar powered sea slug, Elysia chlorotica, practice kleptoplasty in which the slug harvests the chloroplasts from the algae that it feeds on through specialised epithelial cells in the gut. It can maintain these chloroplasts can gain energy from photosynthesis. Genes from these ‘stolen’ chloroplasts only account for 20% of the genes for photosynthesis, and so gene transfer between the algae and the slug takes place via eukaryotic viruses to make up the remainder. Interestingly, these sea slugs typically die once they have laid their eggs and studies have found that this correlates with the appearance of viral particles that were not found in juvenile slugs suggesting dramatic impacts of viruses on life cycles.

Pathogenic or Non-pathogenic

This paper studies the occurrence but more importantly the pathogenicity of three vibrios species on the south coast of Sweden. The species were V. cholerae, V. vulnificus (associated with sepsis) and V. parahaemolyticus (associated with gastroenteritis). The main reason why I like this paper is it doesn’t just concentrate on the occurrence of vibrios like many papers do, but also tries to differentiate between pathogenetic and non-pathogenetic strains.

Their method is very thorough and they used many different methods of which some I have left out. They used mussels as natural filters and collected them from the Sound between Sweden and Denmark over the summer. The occurrence of vibrios was measured by PCR based methods using species-specific and virulent genetic markers coupled with enrichment cultivation methods using TCBS agar. They also performed a cell toxicity test by exposing eukaryotic cell cultures to the isolated vibrios and recording the killing index.

In the PCR based method 53% of samples contained the V. cholerae specific toxR gene of which none were positive for the virulent ctx gene. 63% of samples were positive for the V. vulnificus species specific viuB gene and virulent vvh gene. 79% were positive for the V .parahaemolyticus tlh gene of which 53% were positive for the virulent tdh gene. There were no positive results for any genes when the water temperature was below 17⁰C. In the enrichment method all three vibrios were isolated additionally with V. alginolyticus. These were identified with an API 20NE test kit followed by PCR. All of the isolates were present for the species specific genes stated above but none were positive for any of the tested virulent genes. In the cell toxicity test the killing indices were 78%, 85% and 79% for V. cholerae, V. vulnificus and V. parahaemolyticus respectively.

Overall I thought the paper was really good and the variety of methods used was pretty impressive. They suggest that pathogenic vibrios are common during the summer months possibly even more so than other areas due to the brackish water of the Sound. I found it quite interesting that the virulence genes tested were not present in the isolated vibrios yet they still had a killing index higher than clinical strains. This shows how important it is to use different methods. It also shows that the genes tested aren’t completely necessary for cytotoxicity. Although the cytotoxicity test was using Chinese hamster ovary cells which is very different to the human body and most of the genes tested are suppose to code for haemolysins or toxins which wouldn’t normally affect this sort of cell. The paper shows that the pathogenicity of vibrios is incredibly complex and variable and many different methods should be used to test for it.

A review of: Betty Collin & Ann-Sofi Rehnstam-Holm (2011) Occurrence and potential pathogenesis of Vibrio cholera, Vibrio parahaemolyticus and Vibrio vulnificus on the South Coast of Sweden. FEMS Microbiol Ecol 78: 306–313

Coral species diversity in relation to geographic location- Seminar Paper

For millions of years, a diverse and abundant bacterial relationship has existed with corals. These bacteria can inhabit a range of niches within the corals; however the variation of bacteria between different coral species and their functional role in relation to the host is still waiting to be understood. The idea of mutualistic benefits between bacteria and the host, from processes such as fixation, has been suggest as a reason for this symbiosis. Microbial communities play an important part in coral symbiosis, and have been shown to have the ability to exclude undesirable bacteria by the production of antibiotics environmental changes are resulting in changes in bacterial community dynamics, however the effect of this shift on coral health is unknown. Therefore knowledge of bacterial communities associated with reef-building coral, aids understanding of multispecies mutualism and helps identify which species play a key role in maintaining coral health.

It is thought that bacterial groups may vary between coral species, but evidence has suggested that coral species in different geographic reefs will harbour the same bacterial communities. The aim of this study was to examine coral-associated communities to test assumptions about specificity in coral bacterial associations. Bacterial profiles of three species of Acropora (A.millepora, A. Tenuis, A.valida) from two locations, Magnetic and Orpheus Island, on the Great Barrier Reef, were used to determine whether genetically similar corals differ in structure of bacterial communities. Data collected from the two locations was compared to identify which bacteria were conserved across the geographically distinct locations. An additional study was carried out on A. millepora samples from Orpheus Island to see whether temporal environmental changes would lead to natural variation in coral bacterial communities. The analysis of samples was done using three culture independent 16s rRNA gene profiling methods: clone library construction, DGGE and TRFLP.

DGGE and TRFLP profiles were found to differ between corals from the different reefs, whereas non-metric multidimensional scaling of TRFLP data showed that the samples were grouped according to location rather than coral species. Overall the results indicated that certain bacterial groups associated specifically with corals, but dominant bacterial genera differed between geographically spaced corals. For the additional samples of A. millepora collected, bacterial communities were found to be consistent throughout the year; indicting a stable community despite temporal changes.

A theory proposed for the variation in dominant members of the microbial community in different locations, was the coral probiotic hypothesis: that corals potentially adapt to new environmental conditions by altering species specific symbiotic bacterial partners. This hypothesis seems to make a lot of sense due to evolution; if an organism wants to survive it must adapt to survive in a specific niche. This theory is also backed up by the authors mention that the reefs at Magnetic Island were more coastal and therefore subjected to greater seasonal sea temperature variation, sedimentation and nutrients. Further research should be carried out testing different environmental factors on the bacterial community composition to see how they are affected.

Could a different way of thinking be some corals saviour?

As we are all aware, the increasing frequency and severity of bleaching events and pathogen invasion is contributing to the global decline of corals and many studies focus on microbe communities as the main defense. For example, my group’s paper from the coral seminar (Mao-Jones et al. 2010) doomed corals to a life under pathogenic rule following thermal stress, yet did not consider how the coral host defends itself against pathogen attack. This paper however, focuses on the corals own immunity to disease and the mechanisms it employs, rather than assuming that it’s all down to epidermal microbes.

Importance is placed on this immunity as coral species appear to be declining at differing rates. The paper looks to species-specific immunological responses as the explanation but also mentions that in many cases symbiont differences are used as an explanation as zooxanthellae diversity allows for differences in tolerance to pathogen, thermal and UV stress. However, I tend to agree with this paper as immunity differences would provide much more variation in coral survivorship at species level than the differences between 4 species of zooxanthellae.

The recent discovery of corals possessing invertebrate-like immune mechanisms allowed 4 parameters to be used in this study to determine baseline immunity in several coral species. By comparing the immunity levels between species like this, we can enhance our understanding of the immune pathways utilised to maintain coral health, which could allow us to better predict reef health through environmental changes both in the short and long term, and to reduce those effects by enhancing coral immunity.

The findings suggest, importantly, that the parameters used are accurate measures of immunity as significant positive correlations were found between immunity and susceptibility to both bleaching and disease. However, they also seem flawed as to me; a positive correlation suggests that a higher level of immunity would correlate to a higher level of susceptibility. Although, in the discussion they go on to talk about the inverse correlations of these factors, so it seems unclear as to what they mean. From these correlations though, the authors were able to determine which coral species would be most threatened by a bleaching/disease event.

This finding has important implications as it suggests that some species have naturally have less chance of surviving an event, which leads us to infer that some corals may be predisposed to bleaching or disease. The authors explain this in terms of trade-offs, whereby some corals invest the majority of their energy in other areas of life history than immunity, meaning that they are lacking in this department. Conversely, other corals invest highly in immunity and so are lacking in other departments, e.g. reproduction, but at least survive disease better.

In my opinion this idea and way of thinking makes so much sense and could allow us to manage the issue of bleaching and disease much more efficiently. It would allow us to target specific species rather than managing corals generally, consequently allowing us to put more resources into the species that are most likely to need help. Interestingly, melanin levels had the most influence on coral immunity which could be attributed to its antimicrobial activity and photoprotection properties. This could be an area of research which could help us develop ways to protect corals and their diversity better.

A review of: Caroline V. Palmer, John C. Bythell and Bette L. Willis (2010) Levels of immunity parameters underpin bleaching and disease susceptibility of coral reefs The FASEB Journal Vol.24 1935-1946

Does the Immune Response in Sea Fans Provide Hope for the Survival of Corals during Climate Change?

Climate change is resulting in higher sea temperatures, influencing coral health which leads to an increase in disease. Sea fans become infected with what seem to be opportunistic pathogens such as Aspergillus fumigatuus and A. sydowii, the latter is investigated here. The sea fan speciesGorgonia ventalina is experiencing a prolonged and widespread outbreak of A. sydowii and so is the species of choice in this paper. Immunity to pathogens which take advantage of warmer sea temperatures is seen in many corals but the mechanisms used are not well known. Therefore immune responses, including increase density of ameobocytes and melanization via the prophenoloxidase cascade, are reviewed with particular emphasis on the link between the cellular response and natural warming events (the 2005 Caribbean Bleaching Event).

A. Sydowii is a deep tissue infection which causes darkened lesions on Sea Fans caused by pigmented calcium carbonate. However amoebocytes have been shown to play a role in wound repair, not only in corals but sea anemones also, with influxes becoming known as inflammatory responses because their increase due to infection is so dramatic. Histological images of the mesoglea (connective tissue) allow calculations to be made referring to increased density of amoebocytes here. On average in a healthy coral amoebocytes occupy 15.2% of the mesoglea, in an infected coral this number rose to 24.5%. However in lab experiments this increase is localized to within 1mm of the infection with no difference between corals in amoebocyte density outside of this zone (resulting in the conclusion that there was no systemic increase with regards to fungal exposure). Staining with hematoxylin and eosin revealed that melanin is produced by the host and is found surrounding the infected areas of infected corals. Melanin is a dark pigment that is known to be involved in coral protection, with theories suggesting it keeps UV light away from important symbiotic bacteria. Some amoebocytes also contained black stained granules when used with the Fontana – Masson procedure but this was not consistent for all suggesting differences in immune response function between groups of amoebocytes. Melanin in the form of melanosomes was also noted as present and their synthesis was down to a prophenoloxidase cascade event, an immune response seen only before, in invertebrates.

Interestingly, the sea fans that had been affected by the 2005 bleaching event, caused by the biggest flux of sea temperature in 100 years, showed an increase in amoebocyte density throughout the entirety of the coral tissue, not just in damaged tissue. Different to the results found in corals exposed to the fungus at ambient sea temperature. Replication experiments completed in the lab further supported these findings as keeping corals at a temperature of 31.5˚C for 8 days, compared to those kept at ambient sea temperature, resulted in increased amoebocyte density that was found to be consistent throughout the coral tissue (from 16.9% to 29.2%). This is the first piece of evidence producing results that allow us to see a systemic reaction to elevated temperature.

Amoebocytes, the prophenoloxidase cascade events and the production of melanin have all been observed in Gorgonia ventlina as immune response and resistance to temperature induced fungal infection. The amoebocyte increase is correlated with anti fungal metabolites, immune reactive enzymes and the production of melanin which could provide this coral with an advantage over its scleractinian relatives, which show no amoebocyte increase when faced with fungal infection. The melanin provides a barrier which stops the spread of the damaging A. sydowii fungus. However no long term experiments were reported in this paper with no suggestion as to what would happen if temperature increase was prolonged for longer than what was seen in 2005. Also no systemic system was observed and so the mechanism for the production of amoebocytes located at the points of fungal infection are unknwn and further experiments need to be undertaken.

A review of: Mydlarz LD, Holthouse SF, Peters EC, Harvell CD (2008) Cellular Responses in Sea Fan Corals: Granular Amoebocytes React to Pathogen and Climate Stressors. PLoS ONE

Novel drugs for emerging diseases

A review of: Darabpour E, Ardakani MR, Motamedi H, Ronagh MT (2011) Isolation of a broad spectrum antibiotic producer bacterium, Pseudoalteromonas piscicida PG-02, from the Persian Gulf. Bangladesh Journal of Pharmacology, 6: 74-83.

The marine environment encompasses 71% of the planet and is an ecosystem with a plethora of biological diversity. The spread of emerging bacterial diseases and multi-drug resistance is a serious global concern hence the development of new synthetic drugs and the ‘hunt’ for novel drugs from natural sources are of paramount importance to pharmaceutical companies.

Marine bacteria thrive in niches that require unique metabolic pathways and secondary metabolites to maintain biological activity, consequently opening up numerous possibilities for novel pharmaceutical agents. Several antibiotics have hitherto been isolated from various marine bacteria such as Streptomyces, Alteromonas, Bacillus, Pseudomonas and Pseudoalteromonas. The objective of the study conducted by Darabpour et al (2011) was to isolate bacteria from sediment and water samples taken from various locations around the Persian Gulf, and to test for antimicrobial properties.

Pure colonies of bacterial isolates were screened for antibiotics and tested using several pathogenic bacteria, including multiple drug resistant strains of Bacillus cereus, Proteus mirabilis, Listeria monocytogenes, Bacillus anthracis and Methicillin Resistant Staphylococcus aureus (MRSA). Out of the 48 bacterial isolates, one strain (PG-02) exhibited antagonistic activity to 81% of the pathogens except L. monocytogenes, P. mirabilis and Kliebsiella pneumoniae. PG-02 was identified as a gram-negative, motile aerobe and a Basic Local Alignment Search Tool (BLAST) search in the GenBank database revealed 98% homology to Psuedoalteromonas piscicida strain B14. The inability to use NH₃ and the ability to reduce NO₃ suggests strain PG-02 is a novel species of Pseudoalteromonas, although the authors indicate that this needs to be confirmed by further phenotypic characterization and DNA hybridization.

Antibiotic production began in the middle stages of stationary phase and extended into the death phase, therefore the authors deduce that the antibiotic is a secondary metabolite associated with biomass production. The antibiotic has a bacteriostatic and bacteriocidal effect to a broad spectrum of bacteria harmful to humans, including a number of multiple drug resistant bacteria such as MRSA - a nosocomial pathogen that has increased in occurrence in recent years, particularly in the developing world.

The marine environment is crucial in various global processes and the study highlights other possible contributions that can be obtained from an environment we know so little about. Paradoxically, using novel synthetic and natural antibiotics to treat the aforementioned pathogens may further complicate curtailment because a strong selection pressure accelerates pathogenic evolution and bacteria are able to acquire and exchange genetic information, subsequently giving rise to increased drug resistance.

Microbial Symbionts in Marine Sponges

Our oceans are rich with biological and chemical diversity a large amount of which is still waiting to be discovered. From relatively limited knowledge of marine plants, animals and microbes we have already identified more than 12,000 novel chemicals. The possibilities of us finding more useful chemicals for things like: pharmaceuticals, cosmetics and enzymes are very promising. Sponges are well known to harbour diverse microbial communities and represent a significant source of bioactive compounds. Recent studies of the microbial communities associated with marine sponges have identified a number of new species and an array of new chemical compounds.

Sponges are among the most primitive of multicellular animals. There around approximately 15,000 species of sponge (phylum Porifera) found in our marine environments. Sponges filter particles from water as their primary source for nutrients. They are so abundant that collectively the amount of water they filter is considered to have great ecological importance. They exhibit a vast array of secondary metabolites to carry out functions for example deterring predators, communication and protection against infection. Some of these secondary metabolites have been identified to have anticancer properties making them the subject of much research.

It has often been believed that many products from larger organisms are actually derived from microbial sources. Recent research has identified significant similarities between hosts and their associated microorganisms. Bacteria-sponge associations present a promising source for bioactive compounds. One of the most significant problems that have hindered the search for secondary metabolites is their low concentration. In marine invertebrates many compounds are only found at concentrations of less than 0.00001% of the body weight. So understandable they are very hard to isolate and identify. Compounds that we have already sourced from sponges are suspected to actually be produced by bacterial symbionts due to the resemblance of known bacterial products. This presents very exciting possibilities for large scale production of these compounds because they will be much easier to cultivate then sponges. The symbionts could be cultured or the biosynthetic genes could be transferred into culturable bacteria.

The paper has very limited detailed information. It primarily made up of broad statements which are, in many cases, not backed up with facts, figures or references. However the paper does introduce a very interesting field of marine microbe research highlighting the exciting possibilities for natural products being exploited for our benefit.

A review of: Agus Sabdono. (2008). Microbial Symbionts in Marine Sponges: marine natural product factory . Journal of Costal Development. 11 (2), 57-61