Monday, 9 December 2013

Antimicrobial activity of bacteria isolated from haemolymph of bivalve holobionts

 The lens of the hologenome concept does not see the host and associated microbiota as separate entities, but rather as a single evolutionary unit, the holobiont. The principles underlying this idea lie in how all multicellular life has symbiotes, transmission of symbiotes, benefits of symbioses and how microbiota changes enhance the plasticity of the holobiont against environmental stress.

Bivalves are an ideal model for how microbiota protect marine invertebrate holobionts by competing with pathogens and producing antimicrobial compounds, because filter feeding exposes bivalves to many microbes. The microbiota of healthy bivalve haemolymph has been found to produce antimicrobials and could play an important role in protecting bivalve holobionts from infection by pathogens. This study investigated the antimicrobial activity of culturable bacteria isolated from the haemolymph of an oyster, clam, mussel and scallop species; bacteria with the strongest antimicrobial activity were also tested for cytotoxicity towards bivalve immune system phagocyte cells (hemocytes) and resistance to antibiotics commonly used in aquaculture.

On marine agar, haemolymph bacteria (HB) isolated from the more mobile bivalves Pecten maximus and Tapes rhomboides were almost below detection levels more frequently than those from the fixed bivalves Crassostrea gigas and Mytilus edulis. On average M. edulis had the highest HB concentrations and P. maximus the lowest. The paper posits that HB densities are individual and species dependent, possibly influenced by environmental factors. 843 HB strains in total were isolated from the bivalves and 26 created inhibition zones for at least one pathogen strain; most affected were Vibrio. This apparently differs from most antibacterial spectra, which are never as narrow as this; the specificity of HB antimicrobial activity suggests that the holobionts containing them have been selected because they inhibit common pathogens of bivalves. Other findings for potential further research include the lack of antimicrobial activity in any clam isolates and the rapid loss of HB viability and activity.

Anti Vibrio activity has potential applications in aquaculture, since this genus is a prolific pathogen of fish, molluscs and crustaceans. Oyster and mussel isolates had the most potent antimicrobial activity and were identified to be either the Vibrionales or Alteromonadales orders within the Gammaproteobacteria; nine strains were from Pseudoalteromonas, a genus known to produce bioactive secondary metabolites and increase survival in bivalves. However the strains isolated in this study are phylogenetically distinct from those already used in probiotics and have unique properties.

Hemocytes are short lived and die quickly when incubated in sterile seawater, but the presence of HB strains, their mortality actually decreased and some did in a concentration dependent manner. Reduction of hemocyte mortality by microbes has never been shown before and it demonstrates the role of HB in stimulating the immune system of marine invertebrates. Many HB were found to be resistant to tetracycline, the most common antibiotic in aquaculture, which could apparently be useful for picking out individual strains in future experiments.

This is only a preliminary in vivo study, so it seems to me that the extrapolating HB abundance to be species or fixed/mobile dependent is flawed, given that only four, very phylogenetically distinct species were used, leaving the results open to confounded by evolutionary differences. Future work should also look at how bivalves acquire their symbionts, as it would be interesting if they were acquiring them by filter feeding, the same activity that exposes them to pathogens. I am not aware of how they administer probiotics to bivalves in aquaculture, but it seems questionable that HB will necessarily be able to survive in the guts of other aquaculture species which obtain probiotics orally. The signalling pathways occurring between the HB and hemocytes should be investigated further, because it could represent a good model for how microbes and immune systems communicate with each other; for example, how do hemocytes recognise HB from pathogens? Are there pathogens which imitate beneficial bacteria to avoid phagocytosis?

Desriac, F., Le Chevalier, P., Brillet, B., Leguerinel, I., Thuillier, B., Paillard, C., & Fleury, Y. (2013). Exploring the hologenome concept in marine bivalvia: haemolymph microbiota as a pertinent source of probiotics for aquaculture. FEMS Microbiology Letters.


  1. Hi Dean,
    this is a very interesting study, and more evidence for the hologenome theory of evolution - the HB protect against the most common pathogens in the bivalves, and the microbiota clearly had an influence on the bivalves' health and fitness. It also sounds very similar to the Coral Probiotic Hypothesis where the symbionts were also selected for the most advantageous bacteria; may be they should rename it ;-).
    I'm not sure if I understood it correctly, but it sounded like they were able to culture the strains that were responsible for the production of antimicrobial metabolites? If so, this is a very impressive outcome considering that so many bacteria are not culturable. Have they mentioned anything about the molecular structures of the metabolites or does this need further study and they have just simply observed the inhibition of Vibrio growth? I wonder if the chemical pathways inhibiting the Vibrio sp. would look like with these metabolites.

    1. I guess because the hemolymph is well studied they can replicate it fairly well as a growth medium. as far as I understand it's usually too much nutrients in a medium that prevents the culturing of marine microbes, whereas within an organism, as opposed to in pelagic, environments nutrient levels would be relatively high.

  2. It's strange how bivalves do not have an acquired immunity but they must have acquired the ability to recognize and consequently not destroy these beneficial microbes. Especially as they seem to have some pretty broad spectrum antimicrobial peptides themselves such as lysozyme. Do you think that all the adaptation is on the part of the microbes i/e developing resistance in order to 'help' the mussels rather than infect them? I also think this highlights the segregation of microbiology from other mainstream fields of research as, having just written my literature review on bivalve immunity I came across no reference to beneficial microbes at all leading me to think that we're going about this all the wrong way.

    1. To add to yours and Malin's comments- it seems that the principle idea of these invertebrates being able to defend themselves against pathogens without an immune system is not dissimilar to the one discussed in the lecture yesterday about the cellular mechanisms that produce amoebocytes in corals.

      It sounds like bivalves have adapted a system (i.e. cellular mechanisms as found in corals?) to recognise them as non-foreign microorganisms that don't have a detrimental impact on their health, and actually benefit the ones which take in greater densities of HB to form a stronger symbiosis/ holobiont (such as M. edulis compared to P. maximus) and help drive selection towards these particular advantageous species when in an environment abundant with pathogens (although it is not always clear exactly how these mechanisms work in different species). It would be interesting to compare different species which harbour strains that tend to have more pro-biotic properties to those that favour antimicrobial activity as a form of defence.

  3. Hi Dean,
    I agree with Malin's comment.
    This is indeed adding to the list of evidences that reinforce idea of coral probiotic hypothesis (by Reshef et al. 2006) and hologenome theory of evolution (by Rosenberg & Rosenberg 2008).
    Both of them have emphasized "role of associated microbes in protecting their hosts against pathogens by producing antibiotics" in explaining their theories.
    It is well established that coral associated microbes do this, but the paper that you have reviewed also indicates occurrence of this in bivalve molluscs, which is quit interesting.

    In your review, I don't get -
    you have said - "how microbiota changes enhance the plasticity of the holobiont against environmental stress."
    here what do you mean by plasticity? how changes in microbiota are going to influence plasticity of that organism? I doubt if either probiotic hypothesis or hologenome theory have mentioned change in associated microbiota influences plasticity of the organism!
    I am not aware of any study showing changes in associated microbiota affecting/influencing plasticity of the organism. If there is/are such study/studies, I would like to see them.
    OR you just meant changes in microbiota enhance acclimatization, adaptation and fitness of the organism during environmental stress but I misunderstood because you used the word "plasticity"?

    1. I think he means that the microbiota makes the organism itself more plastic, as it allows the organism to flourish in different environmental conditions. Plasticity here doesn't mean a variation in an obvious phenotype of the organism but more on a physiological level if I understood correctly?
      I have reviewed a paper where the symbiotic microorganisms in the vent tube worm show a different response to the availability of nitrogen, which can be interpreted as a plasticity of the bacteria, but I guess that's a slightly different area ;-)

    2. Thank you Malin
      I was trying to make that perception in my head but got confused by the word "plasticity" which obviously means variation among phenotypes.

      Similarly, I've also reviewed a paper on alkane metabolising microbes from hydrothermal vents. The key point of that paper is - it found enzyme redundancies i.e. they found that bacteria have more than one enzymes for metabolising same sort of alkane which may sound like a wasteful (having more than one enzyme for doing the same job)
      They further argued that different enzymes might work as per the differing availability or concentrations of alkanes present in the environment. I am thinking that this idea of enzyme redundancy may directly relate to what you have said about vent microbes and nitrogen availability.