Antifouling Bacteria in Sponges (Aplysina gerardogreeni)

Antifouling in marine environments as big business, but to date few microbes have been screened for this purpose, with fewer yielding extractable compounds. Marine microbes are exposed to myriad of environmental conditions and so are likely to hold many novel compounds, as well as being able too produce differing compounds in different culture conditions, and having a higher output of these compounds than could be gained from other sources (invertebrates, algae). Aguila-Ramirez et al. (2014) examined bacteria of the sponge Aplysina gerardogreeni (from a family well known for harbouring high numbers of associated bacteria) from the Gulf of California to assess its antifouling qualities.

Samples of 5g tissue were collected bimonthly from April for one year. Though the study does not specify from which region the samples were taken, and whether this was consistent across the study. Bacteria were isolated and cultured in soy broth, and tested for antifouling, antimicrobial, antimicroalgael properties. Bacteria that showed the greatest activity were also identified by partial sequencing of 16S rRNA gene fragments, and matched or compared to their closest recorded relative. This latter method is quite exclusionary, as some bacteria may in a  viable non culturable (VNBC) state, or just less efficient, in the soy cultures used – however as this study seems to ultimately be for commercial purposes, perhaps ease of access in regular mediums is part of the attraction.

The results showed Bacillus, Micrococcus, Paracoccus, Pseudobacter, Pseudovibrio, Psychrobacter, Staphylocuccus and Terribacillus to be present in A. gerardogreeni. These bacteria are reflective of it’s the varied habitats it is distributed in, including chemo-heterotrophs, aerobes or facultative anaerobes, with a fermentative or respiratory metabolism. Preforming functions from bioactive metabolites to protect A. geradogreeni from harmful microbes The latter being an antifouling function, which 50% of the bacteria were found to posses.

The most active isolates were closely related to the Bacillus species, which as Auigla-Ramirez pointed out falls in line with other such research involving this family found in different sources (nudibranch, microalage). Bacillus are widely studied for antimicrobial properties and produce a secondary metabolites synthesised n the ribosome which are especially effective against other members of the Bacillus family.

The temporal variation in activity ranged from the highest levels in February to the lowest in October, with Bacillus by far taking up the largest chunk of time. Other antifouling strains likely compensate for times of inactivity. The lack of activity in winter months indicate that the study has missed out cultures, likely due to VNBC, or/and that biofouling organisms associated with A. gerardogreeni are less prevalent at this time in the Gulf of California.

This study demonstrates some the rich treasure trove of tools waiting to be harvested from the ocean, and that those from sponges will hopefully help along antifouling technology. If there was a greater understanding of the likely symbiotic relationship between A. gerardogreeni and the bacteria which inhabit it, and the reasons fir such temporal variation (ecological or physiological), would most definitely help spur this research along. Use of metagenomics in this study would likely show many more antifoulant bacteria that that may no be able to be cultured, and perhaps change the temporal data gathered in this study. Especially following Deans latest post the A. gerardogreeni and the rest of the Aplysina family are likely more diverse than this study suggests.

Aguila-Ramírez, R. N., Hernández-Guerrero, C. J., González-Acosta, B., Id-Daoud, G., Hewitt, S., Pope, J., & Hellio, C. (2014). Antifouling activity of symbiotic bacteria from sponge< i> Aplysina gerardogreeni</i>. International Biodeterioration & Biodegradation, 90, 64-70.

 

Could global warming increase disease in shellfish farms?

Although we currently have a good understanding of how climate change will affect both physical and chemical processes within our oceans, we do not have the same level of knowledge when considering the effects of global warming on microbial agents responsible for disease.  The areas in and around the Irish Sea in particular are an important commercial source for a number of edible shellfish species such as lobster, edible crab and langoustines, and the farming industries in these areas are economically important.  These species are known to be susceptible to disease caused by marine microbes but it is not known to what extent these diseases affect sustainability in many cases, or how future environmental changes could alter these effects.

For diseases like haplosporidiosis, caused by bacteria of the Haplosporidium genus, which effects haemocytes, connective tissue and digestive gland epithelia in crabs and molluscs there is currently very little data, so possible future effects are unknown.  This disease causes high levels of mortality in infected species and this review highlights a definite requirement for research in this area to understand what conditions are required for this bacteria to thrive, or more importantly, not.

Hematodinium spp. are dinoflagellates that are known to be internal parasites affecting the hemolymph of crabs and lobster species, the disease is commonly known as pink crab disease due to the colour displayed in infected individuals.  It was recently discovered that the larvae of the hosts are also prone to infection and that it is not as previously thought, only the adults that can be infected.  This is of particular concern if global warming is to strengthen ocean currents possibly aiding the distribution of larval forms and therefore the parasites they may contain. 

A little more information is available for Vibrio spp. which include both human and shellfish pathogens.  We know that they generally prefer waters with temperatures of 15°C or above and salinities of less than 25ppt, meaning that costal areas and the species that reside there are at particular risk of infection.  The predicted increases in temperature of costal waters in addition to a decrease in salinity caused by an increase in rainfall due to a less stable climate will provide new areas for natural outbreaks.  It is also known that copepods in particular act as a reservoir for species such as V. cholerae and that they could also be expected to increase in numbers due to warming of costal waters.  There have already been cases of increasing sea temperatures being linked to Vibrio outbreaks in areas such as Chile, Peru and the Pacific Northwest of the United States, but again there are huge gaps in our knowledge of how widespread these effects are. 

It is unclear in many of these cases whether changes in environment will cause an overall increase in such diseases or if an increase in one area may be evened out by a decrease somewhere else.  There are certainly opportunities for research into not only the effects of temperature but also salinity and circulation / current patterns.  We must also consider the speed at which marine microbes may be able to adapt and evolve to allow them to survive in these changing conditions, this only increases the requirement to understand what part viruses may or may not play in gene transfer and control of outbreaks.  These problems could keep research facilities busy for many years, but can we get ahead of the game?

Rowley, A. F., Cross, M. E., Culloty, S. C., Lynch, S. A., Mackenzie, C. L., Morgan, E., ... & Malham, S. K. (2014). The potential impact of climate change on the infectious diseases of commercially important shellfish populations in the Irish Sea—a review. ICES Journal of Marine Science: Journal du Conseil, fst234.

Are globetrotting bacteria just hitching a ride or are they helpful?

The skin of any marine animal provides a unique interface between it and the environment.  Microbial communities found on the skin are known to cause disease in many fish and marine mammals, but could they also be an indicator of good health?

A team from Woods Hole has analysed skin samples from 56 Humpback whales, Megaptera novaeangliae.  This species is of particular interest as they are found in all of the worlds oceans, they also have huge annual migrations which will undoubtedly lead to changes in their environment, with temperatures varying between 6-25°C.  Using using 454 pyrosequencing of SSU rRNA genes, 23 major taxonomic groups were found to be associated with the skin of the humpback whales.  The majority of the bacteria found belonged to the Bacteroidetes and Gammaproteobacteria classes, and many skin samples also contained bacteria belonging to the Firmicutes and Alphaproteobacteria classes.  Two genera, Tenacibaculum (Bacteroidetes) and Psychrobacter (Gammaproteobacteria) were found to be abundant within the majority (97%) of whale skin samples, and collectively made up a large portion, 55–75%, of the skin-bacterial community.  Further analysis showed that differences in abundance of the Tenacibaculum and Psychrobacter spp. were linked to the geographic areas from which the samples were taken.   The same two species also significantly differed in abundance between samples taken from whales that were not actively feeding, in the Pacific Ocean and those who were feeding, in the Bering sea and Atlantic ocean. 

The sequences of the Tenacibaculum and Psychrobacter spp. appear to be specific to Humpback whales although they are closely related to sequences from bacteria taken from dolphins.  Although Tenacibaculum spp. have been associated with disease in fish, it is unlikely that this is the case with this particular species due to the abundance in which it is found on the whale skin.  It has been shown in other studies that Tenacibaculum spp. could actually act as a predator species and could have an antifouling effect, actually helping to keep the whales skin clear and healthy.

Of the two bacterial species, Psychrobacter spp. is globally more widespread and is known to be tolerant of a number of different environmental conditions including temperature and salinity.  Other Psychrobacter spp. are known to be able to produce cold shock proteins and it is therefore likely that the species associated with these whales can also manufacture such proteins to protect themselves against the extreme cold conditions experienced during migration. 

Further investigation is required to determine if the feeding state of the whales which cause them to change to catabolic metabolism while in oligotrophic tropical waters can affect the composition of the associated microbial communities.  While in this state there is a decrease in repair of skin cells, and immune response, which could be linked to decrease in wound healing leaving the animals prone to infection.  So could any of the associated bacteria be producing antibiotics that could aid any healing, or could they be predatory and therefore preventing harmful bacteria from taking hold?  It is important to understand how these bacterial species may be beneficial for humpback whales and also to determine if they are could these properties be exploited for human health or anti fouling products?

Apprill, A., Robbins, J., Eren, A. M., Pack, A. A., Reveillaud, J., Mattila, D., ... & Mincer, T. J. (2014). Humpback whale populations share a core skin bacterial community: towards a health index for marine mammals?. PloS one, 9(3), e90785.

Marine Macroalgae: Defence against bacterial pathogens

Macroalgae supports a wide range of organisms, most importantly in temperate marine ecosystems. They act as a source of food; provide substrata to settle on and protective environments for early life history stages for many invertebrates. As well as being habitat formers and primary producers. Recently, there has been a massive decline in overall biodiversity and loss of algal species in coastal marine environments. Evidence has suggested that macroalgae are under threat from diseases caused by bacteria, viruses and fungi, could be a major cause of the decline. An increase in disease is attributed to opportunistic pathogens that can take advantage of an already weakened host. Therefore, scientists have said that with the impacts of global climate change could result in a greater increase in the rate and severity of bacterial disease of marine macroalgae.

Disease phenotypes often include rotting, abnormal tissue development or changes in pigmentation that appear as blotches, spots, rusts or bleaching. Over the past decade, there has been a gradual increase in the reporting of these symptoms. As there is increasing demand for macroalage as food sources and potential biofuels, it is important that they are well studied.

Macroalgae lack a cell-based, adaptive immune response but do have defence capabilities. Bioactive secondary metabolites are used to regulate colonization of bacteria and other epibionts. These algal metabolites can alter the bacterial community by selecting for beneficial bacterial populations. They can also interfere with bacterial communication networks and gene regulation, in particular quorum sensing systems. Additionally, macroalgal associated bacteria shows signs of inhibitory activities against other surface colonizers, allowing bacteria to outcompete and displace other bacteria.

Pathogen-induced defences include specific recognition of bacteria, followed by a series of immune signalling pathways involving oxidised polyunsaturated fatty acids or oxylipins. However, contrasting data suggests that the signalling pathways are different depending on the species. Oxylipin signalling in red algae increases the expression of stress-related genes and triggers oxidative burst activity in kelp.

Virulence traits in bacteria are highly varied which include toxins, adhesion factors, and mechanisms for nutrition acquisition from the host and to evade host immune responses. Another important virulence determinant of bacteria is the ability to detoxify reactive oxygen species released during oxidative burst of algal cells. Marine bacteria commonly harbour antioxidants and are essential for the progressive virulence, especially in Vibrio species.

Host-bacterial interactions are often highly complex and depend on multiple factors including the state of the host, the pathogenic potential of the bacterium and whether there are any environmental stressors affecting the macroalgae. Tolerances of environmental parameters will affect the overall performance of the algae, making it potentially susceptible to microbial pathogens. Therefore, with environmental changes happening as a result of global climate change, the roles in which bacteria play in structuring the future oceans ecosystems needs to be extensively investigated.

This review included many examples of these processes, however mostly done on terrestrial plants which has then been applied to marine algae, which suggests that more research is needed on this topic. As they contribute to primary production in the oceans and therefore are at the bottom of the food web, all organisms will be affected by the decline in macroalgae populations and this is why I think this is an important area of research.

Egan, S., Fernandes, N.D., Kumar, V., Gardiner, M. and Thomas, T. (2014) Bacterial pathogens, virulence mechanism and host defence in marine macroalgae. Environmental Microbiology. 16: 925-938

Hard life for bacteria in the ocean, some more bacterial grazer just identified.

Molecular survey in the ocean have recently decoded new diversity of microorganisms.  Novel lineages within the three domains of life were unveiled opening new evolutionary and ecological avenues. For example in the eukaryotes were found recently (last year) two new lineages: Picozoa and Rappemonads, both at high taxonomic rank. Such new diversity was detected from ribogroups (i.e. 18S rDNA environmental sequences forming monophyletic clades). People working in this field use also additional tool as FISH, single-cell genomics and (when possible) isolation in pure culture.  

In marine planktonic picoeukaryotes molecular survey had highlighted novel lineage within the supergroup of alveolates and stramenopiles, respectively named MALV and MAST (Marine ALVeolates and STramenopeles). This two group encompassed on the average of 32% and 13% of sequences in picoeukaryotes surveys, respectively. In the MAST group were defined 12 group and 7 more were proposed. Most of these group are uncultured and at the moment are available few data on their ecology and physiology. However in some group, using specific oligonucleotide probe with FISH method was possible discover that MAST cell measures 2-5 μm, are heterotrophic and flagellate. And most important, they are active bacterial grazers.

In this study the main aim was reevaluate the phylogeny, diversity and ecology of MAST ribogroups using three indepent 18S rDNA surveys. The authors (1) search in the GenBank database for stramenopile sequences, (2) analyzed data set of 454 pyrotags from European coastal site (including oxygenated seawater, sediment and anoxic plankton) and (3) used a collection of single amplified genome (SAGs) from single-cell protists.

The authors develop a phylogenetic tree that allow them to identify three additional MAST clades. These clades were rare in environmental sequences so that could explain why these were not identified before. Novel diversity was unveiled in Ochrophyta ribogroups (in this sub-phylum are encompassed diatoms and many other macroalgal group as for example brown algae) and one of these five new group (named MOCH-2) was quantitatively important (around 0.8% of pyrotags that means “a lot”). This was a member of the oxic pico- and nanoplankton. From SAGs was possible to identify some of these MOCH as new algal lineage (plastidic cell) while some other heterotrophic (aplastidic). In these work were also presented the most recent stramenopiles phylogenetic tree based on the available 18S rDNA gene sequencing. From this was clear the main division of stramenopiles phylum in two mayor sub-groups: Ochrophyta (all photosynthetic) and basal heterotrophic taxa (comprehensive also of MAST ribogroups). Combining this data with GenBank sequences authors showed that most of this picoeukaryotes are diffused in all the oceans all over the world suggesting the absence of marked geographic barriers. This confirm the supposed capacity of these minute picoeukariotes to exhibit global dispersion. In this survey also was presented the role of the oxygen status as strong barrier for protists colonization and diversification since 7 MAST group were identified as typical of anoxic systems.

In conclusion and in my opinion, the most important result presented in this work is that 11 MAST groups populate heterogeneous assemblage in surface ocean water all around the world. These group are heterotrophic and for some of them was demonstrated active bacterial grazing capacity. These means that a huge number of phylogenetic diverse picoeukariotes every day in the ocean eat bacteria having a great role in the microbial food webs of one of largest biomes on earth. These results open the way on further studies deeper in the ecology and physiology of these large groups of uncultured planktonic characters. Hopefully using other molecular tools such as FISH probes will be possible to visualize directly these great diversity. An open question is what drive and maintains the diversity of these phylogenetically various but apparently functionally redundant  group of bacterial grazers. Each picoeukariote eat a different bacteria?

Massana, R., del Campo, J., Sieracki, M. E., Audic, S., & Logares, R. (2013). Exploring the uncultured microeukaryote majority in the oceans: reevaluation of ribogroups within stramenopiles. The ISME journal.

Does Hg pollution increase risk of infection?

Exposure to heavy metals and infectious disease mortality in harbour porpoises from England and Wales

Have been on the lookout for some papers supporting the theory that pollution may lead to immunosuppression, leading to an increased risk of disease and this is what I found.

The harbour porpoise (Phocoena phocoena) is undergoing a decline in population size in the southern North Sea and English factor. Many factors have been proposed, most importantly (for the purpose of our module) the question of whether environmental pollution is causing immunosuppression, hence increasing the likelihood of infection. As porpoises are at the top of their food chain they are at risk of bio-accumulating substances such as mercury (Hg)

Throughout 1990-1994 the authors carried out a systematic post-mortem investigation of 86 stranded porpoises in order to establish their cause of death. They only included freshly dead or moderately decomposed animals, and measured both the concentration of metals (Hg, Cr, Ni, Cu, Zn, Se, Cd and Pb) and the cause of death (physical trauma or infectious disease, the latter of which was ID’ed via histological, bacteriological or virological examination)

49 porpoises had died due to acute physical trauma, and were healthy at the time of death. These porpoises were used as controls. 37 porpoises died due to infectious diseases caused by bacterial, fungal and viral pathogens. The majority of the porpoises exhibited severe parasitic pneumonia which is commonly associated with a secondary bacterial infection. Seven porpoises died of generalised micro-parasitic infections (6 cases of bacterial septicaemia, 1 case of morbillivirus infection). I would have preferred some more specific information on what species were causing each infection, especially considering the various tests carried out on each body.

Liver concentrations of Hg, Se and Zn were significantly higher in porpoises that died of an infectious disease when compared to ‘control’ animals (those who had perished due to trauma). However Cu, Cr, Ni, Pb and Cd concentrations did not differ between the two groups.

Little is known about the effects of Zn on the marine mammal immune system, but in humans and rodents Zn is essential for the development and functioning of the immune system. The higher Sn concentrations in infected organisms correlated with Hg levels, this is due to the fact that marine mammals have a mechanism to deal with Hg toxicity, which involves an antagonistic interaction between Se and Hg.

Hg concentration did not vary/ was not associated with differences in sex, region found, season/year of death, state of decomposition and storage method although Hg concentration has been previously reported to increase with age, due to long-term bioaccumulation. The authors concluded that ther was a definite correlation between Hg levels and cause of death (infection) although could not prove any causation (See attached Figure).

Overall the authors are aware of their studies limitations, and concluded that they could not reject the hypothesis that ‘Hg exposure may influence health and contribute to mortality’. I believe this study is a good basis with which to continue our research into answering the question of whether Hg exposure/pollution can lead to an increased risk of infection due to a lowered immune system. I was also impressed by their method of study, this is a difficult question to answer due to the problematic nature of getting data (How do you catch a group of live porpoises, test them for infection and take Hg samples?), but by using the information the sea freely offers (stranded porpoises) we can glean information that will help us understand the big picture.

Bennett, P. M., Jepson, P. D., Law, R. J., Jones, B. R., Kuiken, T., Baker, J. R., ... & Kirkwood, J. K. (2001). Exposure to heavy metals and infectious disease mortality in harbour porpoises from England and Wales. Environmental Pollution, 112(1), 33-40.

Evidence in support of a novel symbiotic relationship in a “bottom-dwelling” bivalve.....

Following a taxonomic review in 2012, Oliver described a tiny bivalve mollusc and named it Syssitomya pourtalesiana, owing to its commensal association with the deep-sea echininoid species, Pourtalesia Miranda.  This minute bivalve, measuring up to 4 mm in length, has the enviable habitat choice of living attached to the anal spines of the urchin (Fig. 1).   In this paper, Oliver et al. (2013) describe the apparent bacterial symbiosis that occurs in this species.

Figure 1.  Syssitomya pourtalesiana attached to the anal spine of Pourtelasia miranda.

A food accepting tract is found along the lower edge of the ctenidial gill, with extensive bacteriocyes lining the surfaces.  The bacteriocytes are densely packed with mainly rod shaped bacteria and coccoid cells to a lesser extent, with pili connecting bacteria in places (see fig. 2).  There is also a layer of diverse bacteria on top of the ctenidial bacteriocytes that are not embedded into the cell walls or within the bacteriocytes, but are interspersed by many haemocytes.   The authors suggest that this association between bivalve and bacteria may represent an intermediate stage to an oblgate symbiotic association, where the bacteriocytes do not enclose the bacteria but instead, phagocytosis occurs by roaming haemocytes.

Figure 2. E. Rod-shaped bacteria within bacteriocyte; pili indicated with arrows; a few filamentous bacteria are also visible. F. Hyphomicrobial cells with paddle-like mother cells (arrowed).

The anatomy and stomach contents of the bivalve indicate that S. pourtalesiana is able to filter feed.  In previously described bacterial symbiotic relationships with deep-sea bivalves, the bivalves appear to have an obligate relationship, relying on the bacteria for nutrients.  S. pourtalesiana however, appears to have a mixotrophic strategy, combining filter feeding with bacterial symbiotic sustenance.  Given the preferred positioning of the bivalve at the anal opening of the urchin, the bacterial metabolites are likely to originate from urchin faecal matter.  Many of the filamentous bacterial cells found atop the bacteriocytes are Hyphomonas-like, with paddle shaped swellings at the tips of many (fig 2.).  Hyphomonas bacteria are known to be a primary food source of a hydrothermal vent limpet, Lepetodrilus schrolli and this bacterial genus utilises DOM.  The exterior positioning of the filamentous bacterial cells on the ctenidium indicates that they are unlikely to be a source of food for the bivalve, giving further support to the idea of symbiosis.

One of the key driving forces of chemosymbiosis is the response to nutrient poor conditions, such as those commonly found in the deep ocean.  Furthermore, the presence of an apparently diverse microbial community associated with the bivalve gill may indicate utilisation of a wide range of trophic pathways, as seen in the magnificent gutless worm, Olavius algarvensis.  Unfortunately however, the authors were unable to isolate any of the bacteria or carry out any molecular analyses, limiting the weight that can be attributed to the observations made.  The evidence is compelling however and genomic analyses of the bacteria is imperative to be able to understand the processes involved.   Given the recent taxonomic description of the bivalve and apparent novel symbioses in this family, I hope that follow up work can ascertain the nature of the symbiosis, whether bacteria are acquired or inherited and how well the bivalve can continue with biological processes in the absence of the bacteria.

Oliver, P. G. (2012). Taxonomy of some Galeommatoidea (Mollusca, Bivalvia) associated with deep-sea echinoids: a reassessment of the bivalve genera Axinodon, Verrill & Bush, 1898 and Kelliola Dall, 1899 with descriptions of new genera Syssitomya and Ptilomyax. European Journal of Taxonomy, 12, 1-24.

Oliver, P. G., Southward, E. C., & Dando, P. R. (2013). Bacterial symbiosis in Syssitomya pourtalesiana Oliver, 2012 (Galeommatoidea: Montacutidae), a bivalve commensal with the deep-sea echinoid Pourtalesia. Journal of Molluscan Studies, 79(1), 30-41.