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Flashcards in Biological adhesion in the marine environment Deck (19)
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Historically, much work has been done on Semibalanus balanoides, largely in an ecological context (some classic ecological experiments):

Give some factors affecting settlement


  • Hydrodynamics
  • Available space
  • Topography 
  • Incumbents (e.g. biofilm, algae and conspecifics)
  • Predation, competition and disturbance
  • Colour

More recent studies have focussed to a large extent on Balanus amphitrite, and other fouling species driven by the the need for new antifouling technologies 


What is the difference between cues and signals? 

Signals are involved in conspecific communication whereas cues are between different species. 


What concepts/properties have received most attention with regard to cyprid settlement:

Chemical (biological cues & signals)
Surface energy

(Modulus as been studied almost exclusively with adults)


Tony study - looking at barnacle settlement at two sites 

Found a difference in the settlement behaviour between two sites either side fo Prince Edward Island. One site preferred the crevasses (considered normal behaviour) whereas the other did not have the same preference for crevasses


Settlement of S. balanoides almost exclusively inside 
       > 10 cm crevices 
Settlement more important outside crevices

BUT on a small scale (<1.5 cm deep cracks), the presence of conspecifics had a stronger effect on settlement than heterogeneity in both regions. 


Chemical basis of gregariousness

  • Many species obligate cross-fertilisers meaning they need to mate with a neighbouring barnacle.
  • Hermaphrodites – but need to cross-fertilise
  • Proximity achieved through the gregarious settlement of the cyprid
  • Collect sperm through filter-feeding – probably 
  • Barnacles spread sperm using filter-feeding to clap sperm, requires close proximity


What was the first hint that there was something left behind by the barnacles which is attractive? 

Knight-Jones EW (1953) J Exp Biol 

A paper looking at gregariousness during settling in Balanus balanoides and other barnacles in 1953, at Bangor. 

If barnacles are removed, and new cyprid larvae placed they will settle where the previous barnacles were.



What study and findings followed the results from Bangor 

1962 – a paper produced in Anglesey

Showed that a slate soaked in L: precipitate of partially purified extract of adult barnacles induced barnacle settlement

  • The water-soluble signal that readily adsorbs to surfaces and active in surface-bound conformation
  • Suggested to be associated with the integument of the barnacle – referred to as ‘arthropodin’ (an old term for a water-soluble protein of arthropod cuticle)
  • More active on conspecific compared to allospecific species.
  • Perceived via contact or tactile chemical sense


paper 3 - boiled seawater 

Published from NERC in 1981 – looking at physiochemical properties of the settlement factor proteins 

Test extracts that were native and boiled. Did this as if you boil seawater extracts of barnacles they retain their activity but the process of boiling gives the precipitation of proteins – removing inactive proteins but also breaks down proteins.
Native unboiled extracts - when they did the separation of the proteins on gels – separating them out according to subunits peptide length. The maximum size matched what has currently been found. 


Barnacle settlement cues - japan paper 

What did they look at?

Specifically, the project searched for settlement-inducing and -inhibiting compounds from various sources. It also investigated the expression of the function mediated by second messengers.

Focused on two species - subtropical 

Multiple brooders - bring into the lab with summer conditions and will produce larvae throughout the year - contrast with the work of Crisp on semibalanus over 30 years.

Able to settle in the absence of flow - able to do experiments in petri dishes.

Great lab animals and an important fouling spp. 


Barnacle settlement cues - japan paper 

What did they discover?

Three major sub-units – termed the settlement-inducing protein complex.

  • Separations of extracts, into different fractions and ask the cyprid which fraction do you like? - the essence of the purification strategy.
  • The factor was isolated in its pure form.
  • Something about the subunit composition was determined. 
  • Proteins can be post-translationally modified after production, this protein had sugars added to it. As it had multiple subunits the Japanese called it SIPC. 
  • Made antibodies for all of the subunits allowing the presence of the antibody to be probed.
  • Molecules called lectins are sugar-binding proteins, which have been used to probe glycoproteins to give an idea of which sugars are associated with proteins. 
    • using a range of lectins it was shown that lentil lectin was pretty effective at reducing the settlement (induced by SIPC).
    • Because lentil lectin bound the SIPC it could be used in packing for chromatography columns allowing for a final stage in purification. 
  • The subunits were assayed for activity (76k, 88k and 98k), all the subunits are virtually equally active. Using 76 - shows it was present more or less throughout larval development.
    • important cause of footprints 


Japan study - the identity of the SIPC 


Able to clone and sequence SIPC - can imply amino acid sequence is - giving a molecular mass of around 200 kDs. Closely related to a group of molecules involved in innate immunity - mechanism of action to inactivate proteases  (which are involved in immunity by attacking pathogens.

Significant homology to the highly N-glycosylated α2-macroglobulin (A2M) and insect thioester-containing proteins (TEP). Related molecule but different as the barnacle does not have a thiolester domain. 

Almost all the sugars that bound to the molecule are mannose. N- linked glycans enzymatically liberated from the SIPC were found to be high-mannose-type ranging from M2 to M9.

RNA probe – look where transcription was occurring and staining the epidermis which underlies the cuticle - provided strong evidence that it was a cuticular protein.


How species-specific is SIPC?

  • The SIPC is not species-specific but activity accords with systematic affinity.
  • Differences in the sugars that are bound to the molecule and regions of the molecule diverge. 


Concentration-dependent effect of the SIPC (recombinant)

  • Barnacles settle closely at low concentrations but at higher concentrations settle further away from each other - space to grow and reduced competition. 
  • At lower concentrations of SIPC, as you increase the concentration it became more active at inducing settlement. There becomes a point where this activity fell off. Territorial behaviour of barnacle Cyprus larvae.  


A most recent study - in the US. 

  • Predatory whelk (Acanthinucella spirata) prefers to feed on ’faux’ barnacles containing MULTIFUNCin compared to mussel and turban snail extracts
  • Also active as settlement inducer to conspecific (Balanus glandula) cyprids
  • The first idea that these molecules had multiple purposes - and gained the term keystone molecules in affecting population community dynamics. May deter predators such as Nucella.


Function(s) of the SIPC in the cyprid


  • So there is strong evidence that the SIPC is an adult signal to settlement of cyprids.  But SIPC is not confined to the adult stage.  It is also present throughout larval development.  
  • We also have evidence (next slide) that the more a surface is explored by cyprids, the more it becomes attractive to conspecifics. 
  • This opened up the intriguing possibility that cyprids could communicate to each other via the SIPC – is the SIPC a cyprid signal?
  • Yule & Walker (1985) first proposed that cyprid-cyprid interactions via proteinaceous ‘footprints’ are the primary basis of barnacle gregariousness. SIPC is at least a component of the ‘footprint’ protein and a stciky? protein.



There is strong evidence that gregarious settlement of barnacles has a chemical basis
While there are undoubtedly other factors at play, the settlement-inducing protein complex (SIPC) is an important modulator of gregarious settlement, including cyprid-cyprid interaction, perhaps especially in relation to species recognition
There is preliminary evidence of the involvement of other pheromones, which effect a different response to the SIPC
Interestingly, the SIPC (MULTIFUNCin?) may also be utilised by predators of barnacles


Surface energy and charge

Important if you are going to design a process to inhibit barnacles themselves 

Published in the 1990's respond differently to hydrophobic and hydrophilic surfaces.

Balanus amphitrite cyprid settlement influenced by surface charge 

  • Similar surface energy but:
  • Significantly higher settlement on the negatively-charged ( COO- ) SAM
  •  Intermediate settlement on the neutral  OH SAM
  • Lowest settlement on the positively-charged ( N(CH3 )3+ monolayer.
  • Similaries - easier to produce a surface that inhibits them both 




barnacles have red autoflouresence 

colour but not brightness fo substratum affects settlement 

H - cyprids use the compound eye to see adult barnacles - make sure all coatings are the same colour.