Barnacle Adhesion

Question 1

Why the interest in barnacle adhesion?

Answer 1

Important to understand the process so you can interfere with it in antifoulants.

More environmentally sustainable adhesives - particularly for use in surgery.

Question 2

Barnacle diversity

Answer 2

Arcothoracicia - burrowing barnacle - live in shells and stuff

Thoracica - common acorn and stalked barnacles.

Rhizocephala - parasitic barnacles.

Variety of substrata - live on mammals - crustacea - artificial structure- coral barnacle

Variety of habitats - deep-sea - open ocean - intertidal

Question 3

Overview - barnacle adhesion

Answer 3

• Aim is to examine the variety of adhesives used throughout the barnacle life cycle to maintain contact with a substratum. Although characterisations are incomplete, adaptations according to function are emerging for the ‘different’ adhesives.
• The cyprid ‘temporary’ adhesive – used to explore the surface (D)
• The cyprid cement – used to permanently attach the cyprid to the surface and maintain adhesion until the adult cement is produced (E). Cyprid vulnerable during this time - strong cement.
• The adult cement – produced throughout the life of the adult barnacle (A). As the barnacle grows more adhesive has to be used.

Question 4

Cyrpid attachment organs

Answer 4

Cyprid attachment organs and compound eye (only stage of the life cycle with the compound eye), leftover nauplius eye leftover from the nauplius stage, paired cement glands which produce cement to anchor the cyprid down permanently. Paired antennules – the tip of disc is very hairy – possibly important as they may impart some adhesive characteristics to the cyprid larva.

Question 5

Pre - settlement behaviour

Answer 5

1. Start out by engaging in wide search behaviour and tend to walk in a straight line.
2. Exploring a particular area - anchor down with one antennule which sticks and they probe around with the other using the setae on their antennules
   3.

Question 6

Nature of the footprints - what can be seen in AFM imaging mode?

Answer 6

Atomic form microscopy and protein staining show the adhesive is proteinous.

AFM reveals nanofibrillar material deposited - a common feature of barnacle adhesive.

Question 7

What is SIPC?

Answer 7

Dreanno et al. (2006) Biol. Let

Settlement inducing protein complex which is a pheromone used for cyprids to sense adults.

If a cyprid walks over a Petri dish, and the water is replaced, the petri dish induces greater settlement - pheromone in the adhesive.

Attractiveness can last 2 weeks in natural seawater.

Question 8

What did Nick Aldred discover about barnacle attachment from the University of Copenhagen?

Answer 8

Barnacles do leave footprints behind.

Cyprids able to apply pressure to substratum, spreading out disc increasing contact area, increasing adhesion - behavioural component.

Question 9

What advance was gained by using iSPR?

Answer 9

Imaging surface plasmon resonance (iSPR)

Image material deposited on surfaces - a thin layer of gold.

Cyprids footprints

Question 10

What questions can be asked about SIPC?

Answer 10

If the SIPC serves a temporary adhesive function, it should be a ‘sticky’ protein

For fouling species, the SIPC should also be able to stick to a diverse range of surfaces

Question 11

Can SIPC bind to diverse range of surfaces?

Answer 11

Petrone L et al. (2015) J R Soc Interface

SPR data for the adsorption of fibrinogen, SIPC and α2-macroglobulin (A2M) on a series of SAMs.

SIPC shows the most similarity to fibrinogen rather than A2M. Fibrinogen is a sticky protein.

Question 12

Hybrid wet/dry adhesive system?

Answer 12

• Atomic force microscopy (AFM) of cyprid temporary adhesive
• If you treat the glass with NH2 it binds the cyprids really well.
• Interaction of hydrophilic silicone nitride AFM tips and hydrophobic CH3 tips with Semibalanus balanoides footprint protein
• Mean pull-off force of 0.41±0.20 nN s.d. (N=2500) (Semibalanus balanoides)
• If we assume 50% porosity of footprint and scale force from the area of AFM tip to that of two footprints, estimated protein adhesion strength = 0.026 MPa
• Empirical measure (on glass) = 0.098 MPa (Walker 1987)

Question 13

Hairy discs

Answer 13

Why were the results for the mean pull-off force from the experiment using a wire 0.098 and using AFM - 0.026 MPa?

The high density of villi may contribute to the high strength of adhesion required to resist dislodgement.

Breaking of sacrificial bonds between polypeptide chains = natural damping mechanism?

Sore tooth pattern – noticed when using AFM.
This sort of arrangement is characteristic of pulling of groups of polypeptides which interact with each other, known as sacrificial bonds – break and release energy.

Question 14

Cyprid cement

Answer 14

produced by paired cement glands, released by a single gland which runs down each antennule. The cement gland comprises a number of cells which can be isolated by using an enzyme.

Look in detail with an electron microscope – alpha and beta which contains two size classes of granules – small (beta).

No one knows how the cement is released apart from that it is released very quickly.

Study in Japan shows that the granules are released quickly

Question 15

What proved there being an alpha and beta protein in cyprid cement?

Answer 15

B granule – stain for lipid, Alpha granules stain for protien

Question 16

What is discovered about cyprid cement when looking at the plate in detail? Gohad NV et al. (2014) Nat Commun

Answer 16

When you look at the attachment disc under the microscope

Around the protein core there is a lipid phase

Contents of the beta granules released before alpha granules.

Lipid granules released first - removing water, the apha - which is the adhesive is released second.

Question 17

Localisation of chitin in the beta cells

Answer 17

Looked for and found chitin, at the interface of the protein and the lipid. The other interesting feature is that when you look at the beta granules- chitin is associated with the membrane of the vesicle.

Question 18

Is chitin a functional component of the cyprid cement?

Answer 18

Adding chitinase to a dish with attached cyprids before metamorphosis it will eat away at the cement and release the cyprid. After metamorphosis when the permanent cement is produced the juveniles are not removed (chitinase probably can’t gain access)

Seems to be a functional role of chitin.

Reading would benefit grants

Question 19

The adult adhesive system

Answer 19

Needs to be deposited as the barnacle grows.

Primary ducts extend from the initial point of attachment and is replaced with each moult.

1 important aspect of the cement. It has a fibrous structure, the mass spectrum shows a peak amide and amyloid. suggesting amyloid fibres - are an important component of the cyprid cement. Can also image using a florescent stain.

Question 20

Phase separating fluid - adult cement

Answer 20

Clears the water prior to adhesive being deposited

Phase separating fluid rich in lipids and reactive oxygen species to clear surface ahead of cement deposition

Question 21

What is known about adult barnacle cement?

Answer 21

Silk like motifs - So et al. (2016) by transcriptome-led proteome sequencing of Balanus amphitrite have described ~50 proteins, many of which are previously unidentified and comprise a significant component of barnacle cement – they are of low complexity, have blocks of repetitive sequence with homology to silk motifs

Question 22

Summary

Answer 22

The adhesives of barnacles have been the subject of intensive research over the past decade
Most progress has been made on adult cement. Although far from complete, a picture is emerging of a highly complex system of proteins and other components, specialized to displace water and contaminants and to stick to a variety of surfaces, with release coordinated with barnacle growth
The cyprid, described by Crisp as the “pinnacle of sessile evolution”, has received comparatively little attention. The ‘temporary adhesive’ is specialised for reversible adhesion as the cyprid explores surfaces and for resisting hydrodynamic forces; one of its components is SIPC-like. In common with the adult cement, the cyprid cement is a multicomponent system. The two cements appear to contain some related elements. Moreover, the cyprid cement has the ability to displace water with a lipidaceous secretion prior to release of the proteinaceous ‘adhesive’
Only a handful of fouling species have been studied. Given the diversity of barnacles and habitats that they colonise, there remains much to learn!