Principles of Barnacle Adhesion

Question 1

• Ways of sticking to surfaces:-

Answer 1

• Mechanical
• Suction
• Viscosity
• Capillarity
• Friction

Question 2

What is adhesion?

Answer 2

“The action or process of adhering to a surface or object”

Question 3

Why is adhesion more common in the marine environment?

Answer 3

Unlike on land, marine animals can be sessile because food is brought to them, waste is taken away and gametes dispersed.

This saves energy, but their attachment must be strong as development causes mortality.

Question 4

Synthetic adhesives

Answer 4

• sticking underwater
• does not require clean surfaces

Question 5

Mechanical interlocking

Answer 5

Mechanical interlocking - lock and key type adhesion to the surface (although electrostatic interactions are also required…).
• At the micro-scale, most materials are rough.
• The glue must be able to spread on the surface.
• The glue must be able to enter the roughness features.
• The glue must then harden.
• All of these processes present challenges underwater, as we will see!!

Question 6

Mechanisms of adhesion:
2. Suction & Stefan adhesion

Answer 6

Neither of these is really ‘adhesion’ mechanisms per se, in that there are no attractive forces. Rather, they are physical processes that interfere with removal.

Suction

• Requires an elastic cup which is initially contracted and then expanded to form a zone of reduced pressure.
• Not attractive.
• Theoretical maximum resistance of 1 atm.
• Very weak in ‘shear’. Easy to slide off.

Stefan adhesion

• Resistance to viscous flow
• No zone of reduced pressure
• Not attraction
• Attachment pad - viscosity of medium and speed the pad is being moved

Question 7

Capillary tubing

Answer 7

• Depends on the surface energy (determined by the chemistry) of the surface and the surface energy (called surface tension…) of the liquid.
• If the tube has more ‘energy’ (is more reactive) than the liquid, the liquid will ‘prefer’ to stick to the glass, rather than to itself, so it is drawn up the tube. Glass has a lot of free energy compared to water.
• Can also draw surfaces together, but liquid must have lower energy than the surface for this to work.
• But like suction, capillary is very weak in shear.

Question 8

Friction

Answer 8

Octopus (among other things) - manage to attach very strongly using, ostensibly, a suction mechanism. - stronger adhesion than 1 atm

• This supernatural feat, and many others, can be ascribed to a frictional contribution to adhesion.
• In order to break a suction bond, the seal must be compromised – usually by pulling inwards.
• If this can be prevented, the suction force is theoretically limitless.
• Slip of the perimeter seal is governed by friction….

Question 9

Many different unrelated organisms have developed similar ways in overcoming adhesion problems - give the example of tree frogs.

Answer 9

• Tree frogs secrete a liquid adhesive (mucous) from their soft toe-pads, producing a capillary effect
• Capillary adhesion is weak in the ‘shear’ direction, however, so frogs must also use boundary-layer friction between their cuticle and the surface to prevent slip.
• Toe pads subdivided into little grippers

Question 10

Many different unrelated organisms have developed similar ways in overcoming adhesion problems - give the example of bush cricket.

Answer 10

The tarsi of the cricket Tettigonia viridissima also have smooth flexible pads that are used for capillary attachment.
A liquid (cuticular oil) is also secreted from between the lamellae of these pads, mediating adhesion in a similar way to tree-frogs.
Microscopic droplets of water in the cuticular oil form an emulsion that stiffens under strain. The more water, the more resistance to shear.
This ‘non-Newtonian fluid’ hardens ONLY when a force is applied!
J. R. Soc. Interface., 2006; 3:689-697

Question 11

Challenges od adhesion mechanisms for marine organisms

Answer 11

• Surface energy
• Surface hydration
• Surface contamination
• Surface roughness

Question 12

Describe surface energy

Answer 12

• Surface energy is a measure of the ‘reactivity’ of a surface; how capable it is of forming new bonds with materials it comes into contact with.
• Hydrophilic - high energy - love water - water spreads out across the surface.
• Hydrophobic - low energy - hate water - water forms droplets
• Superhydrophobic - specific surface texture means water barely even comes in contact with it.

Question 13

Challenges - surface energy

Answer 13

Glue gets outcompeted by water for contact with the substrate

what is true in air does not translate underwater….

• A hydrophilic surface, which would be optimal for adhesion in air, can be very difficult to contact underwater due to strongly bound water – a hydration layer.
• Ironically, therefore, organisms may be expected to adhere better to low-energy surfaces underwater better than they can to high-energy surfaces.
• Some organisms settle with sticking to hydrophobic surfaces that they can contact easily. But some either remove water from the interface, or incorporate it into their glue, so that they can stick strongly to hydrophilic surfaces. •

Question 14

Challenges: 2. Surface hydration

Answer 14

Removing water from the interface:

• • Mussels and barnacle larvae (cyprids) both use lipids to remove water from surfaces which, presumably, enables them to attach more easily to immersed hydrophobic surfaces.
  Incorporating water into the adhesive:
  • Water can be incorporated into adhesives in a process known as ‘complex coacervation’, mussels do this
• Recognise that a high energy surface is the best to stick to, so remove the water

Question 15

Challenges: 2. Surface hydration

Question 16

Challenges: 3. Surface contamination

Answer 16

Research does not have a good handle on how organisms deal with surface contamination.

• Just as was the case for dealing with water, biofilm, for example, can be either incorporated into the glue, or removed from the surface.
• It seems that the tubeworm Hydroides may do the former, while mussels appear to brush away biofilm with their foot before attaching.
• Surface contamination also, inevitably, affects the surface energy and the wetting properties of the surface.

Question 17

how does surface Surface roughness affect adhesion

Answer 17

• Generally speaking, rough surfaces have stronger adhesion (when mechanical interlocking is used).

however, whether the glue penetrates the texture depends on whether the surface is in the Cassie or Wenzel state…

which, in turn, depends on the surface energy of the material.

tend to find the cassie state on superhydrophobic surfaces that has been roughened in a certain way.
• If the flat material has a contact angle when its smooth of more >90o, then it may enter the Cassie state when rough it becomes more hydrophilic.

If the flat material has a contact angle of less than 90o, when it becomes rough it becomes more hydrophilic, wayter runs through nooks and cranny and spreads out.

Question 18

What are the different states a water droplet can be in?

Answer 18

Water droplets on rugged hydrophobic surfaces typically exhibit one of the following two states:

(i) the Wenzel state in which water droplets are in full contact with the rugged surface (referred to as the wetted contact) or
(ii) the Cassie state in which water droplets are in contact with peaks of the rugged surface as well as the “air pockets” trapped between surface grooves (the composite contact)

Question 19

Challenge 4 : surface roughness

Answer 19

• Remember, all of this changes underwater….
• A surface in the Cassie state may retain trapped air when placed under water which ‘may’ (the jury is out on this…) help prevent adhesion.
• If the air is not trapped, however, even a textured hydrophobic surface will enter the Wenzel state when placed underwater.
• This will allow entry of the adhesive into the texture and promote strong adhesion.

Question 20

Challenges 5 reversibly

• describe tube feet

Answer 20

• Not many adhesive mechanisms are truly reversible.
• One that certainly is not, but looks like it is, is the tubefoot of the seastar….
• Involves a 3-gland system.
  
  • Sfp1, (starfish foot protien 1) a large protein of 3853 aa, is the second most abundant constituent of the adhesive.
  • Sfp1 is translated from a single mRNA and then cleaved into four subunits before being linked back together by disulphide bridges in a different combination - mature adhesive protein - not sure why it does this.
• Present in one of the glands and secreted and mixed with the contents of another one of these glands and produces a very strong teo component permandent adhesive bond.
• releases an enzyme from a third gald, digests the adhesive at the interface - brief permanent adhesion mechanims

Question 21

Suction remora

Answer 21

The remora’s ‘sucker’ is a modified dorsal fin.

The fin is flattened into a pad and surrounded by a thick lip of connective tissue that creates the suction seal.

The lip encloses rows of plate-like structures called lamellae, from which rows of tooth-like spinules emerge for mechanical grip.

Question 22

Suction Cephalopods

Answer 22

In the cephalopods, use of a liquid glue is an ancestral trait. More advanced cephalopods rely more on suction.

4 genera (Nautilus sp., Sepia sp., Euprymna sp. and Idiosepius sp.) use liquid adhesives.

One genus, Euprymna, also uses a deadhesive – i.e. produces one material to attach and another to detach!

Question 23

Limpets

Answer 23

• Modern-day limpets also rely on suction for adhesion, using it for attachment during locomotion in addition to capillary/Stefan adhesion.

When exposed by the tide, however, limpets secrete a true adhesive hydrogel, containing:

o <97% water and several polar proteins,

o A 140 kDa glycoprotein complex for adhesion,

o Gives a tenacity of <500 kPa.

(all that’s known about marine gastropods)

Question 24

snake and slug

Answer 24

• Similarly, the terrestrial slug Arion subfuscus produces a defensive secretion that is sticky and tough, despite being more than 95% water.

In tensile tests, the glue sustains an average peak stress of 101 kPa (it is strong!), and fails at an average strain of 9.5 (and stretchy!). How??

Question 25

• What are some key constituents of slug hydrogels - . If calcium, magnesium and iron are removed the glue experiences a 15-fold decrease in storage modulus.
• Proteins and carbohydrates have a negative charge but also crosslink positively charged sugars, stored separately in structures.
• Metals act as counter ions to keep everything electrostatically neutral. When the stuff is secreted it is all rearranges and the metals crosslink the proteins and the carbohydrates but they do not carbohydrates to the protiens - slug glue - double network.
  
  • A network of protiens crosslinked by metals
  • A network of carbohydrates crosslinked by metals - interspersed by separate.
• Carbohydrate - runs through the protein network and is not stretchy - pull-on glue you break carbohydrate bonds but it is held together by strchy protien.
• Protein - stretchy held together by metals - on its own

Question 26

Adhesion of mussels: The byssus

Answer 26

• Mussels produce a byssus of adhesive threads
• These threads are formed along the ventral groove of the foot in a process resembling injection moulding.
• There are three major glands: the phenol, collagen and accessory glands. These feed specific amounts of their contents into the ventral groove to form the adhesive plaque, the collagenous (similar to a tendon) thread and the cuticle of the byssus.
• Those proteins forming the adhesive bond of the plaque to the surface are deposited first at the distal tip of the foot, followed by the bulk of the plaque and thread core components.
• Finally, just before the thread disengages from the groove, the structure is coated by a 5-μm-thick cuticle from the accessory gland.
• The byssus has as many as 20 different known protein components

Question 27

Adhesion of mussels: Composition

Answer 27

• The mussel foot proteins (important proteins for sticking) (Mfp)-2, -3, -4 and -5, originate from the phenol gland and are destined for the plaque.
• Mfp-1 (Mytilus foot protein) and Mfp-6 have been localized to the accessory gland.
• Collagen gland proteins, such as the prepolymerized collagens (preCOLs),
  
  • 3 types with distal (D), proximal (P) and nongradient (NG) distributions (preCOL-D, -P and -NG) are used in the thread core.
• Although Mfps do exhibit some chemical diversity, most are glycine rich and contain DOPA, and all are moderately to strongly cationic.
  
  • DOPA is a post-translational modification of tyrosine and by far the most well-studied adhesive molecule in biological systems. DOPA is used as a precusor for melain etc in a human. Mussel take a chemical and uses it for something completely different.

Question 28

Steps of adhesion mussels

J. Exp. Biol., 2017; 220: 517-530

Answer 28

1. Clean biofilm and mess that may interfere with adhesion
2. Foot extended
3. Cavitation
4. pH adjustment - produces an acidic environment - DOPA is pH sensitive
5. Redox adjustment - introduces negative charges in the form of electrons into the distal depression
6. protein secretion - once happy with the pH and redox conditions it will start producing protiens
7. Coacervation
8. Phase Inversion
9. Complete assembly
10. Solidification

Question 29

What happens in the two pH conditions whilst a mussel is sticking the collagen thread to the substrate?

PH4 or lower

Answer 29

pH4 or lower

• DOPA is in its reduced form, has OH- groups on it, it’s phenolic (has a phenyl ring)
• Mussel foot proteins can be up to 30 - 40 % - huge amino acid bias.
• OH- groups can interact strongly with mineralised surfaces (such as iron in rocks)
• Thought that mussels use another amino acid - lycine which is extremely hydrophilic and interspersed with DOPA. The theory is that lycine gets the adhesive protein to the surface, once its at the surface the DOPA molecules adhere.
• After this, the mussel lifts its foot the surface and the collagen thread has adhered but not yet cured.

Question 30

What happens in the two pH conditions whilst a mussel is sticking the collagen thread to the substrate?

ambient seawater

Answer 30

To cure the pH has to return to the ambient pH of seawater (8.2).

• at 8.2 DOPA behaves very differently at the ambient pH
• DOPA is no longer in is reduced form, OH- ions pinch the H+ ions of the DOPA leaving two oxygens attached to the phenyl ring, called a dopaquinone. Dopaquinone is very good at sticking together, so links up all the unattached oxygens forming the solid mass that is stuck to the surface.

Question 31

What is coacervation?

Answer 31

• Where you have an emulsion of different charges, which can phase separate
• The different proteins which are secreted into the mussel glue do not mix completely and exist phase-separated like droplets inside another liquid.
• At ambient pH all the pockets get trapped and its like a sponge or solid foam.
  
  • This has mechanical advantages, as energy is lost each time you hit solid material.
  • Disperses energy like the double network in the slug glue.
    *

Question 32

What is the redundancy in chemistry that mussels show?

Answer 32

Mussels dont rely on 1 adhesive mechanism, they have

• mechanical strength from the porous material
• redundancy in the types of chemistry they use to attach to surfaces

​Interfacial interactions

• H-bond
• Electrostatic
• hydrophobic

Cohesive interactions

• Electrostatic
• Hydrophobic
• H-bond

Question 33

Mechanical properties of the byssal thread

Answer 33

The network of threads efficiently redistributes load.

• The treads have self-healing abilities if they are stretched beyond their elastic limit.
• The pre-Cols of the thread are arranged in an elastic gradient to spread the force away from the animal. Avoids a weak point in between tough and soft tissues
• The plaque is porous to enable ‘crack trapping.

Question 34

What is the second best-studied marine example in terms of adhesion?

Answer 34

Adhesion of Phragmatopoma: Pacific species

‘Sandcastle’ worms glue particles together to form a tube.

• Although the application of their adhesive is very different to mussels and echinoderms, there are mechanistic similarities.
• Rather than a ‘multi-gland’ system producing a complex structure (mussels), the worms use a ‘duo-gland’ system to produce a liquid glue that hardens underwater

Question 35

Phragmatopoma

similarities to slug and mussel?

Answer 35

they have a number of types of gland which they store the adhesive precursor in

They have adhesive proteins, which have a large amount of DOPA and phosphorylated serine which do similar thing to they do in the mussel system despite them evolving independently.

They also use complex coacervation’.

In the two adhesive glands, they also have lots of metal ions to balance the charges.

Question 36

Adhesion of Phragmatopoma: how does it work

Answer 36

• Two gland types with different proteins and counterions (calcium and magnesium).
• These are released to the building organs which is a sack it holds by its mouth - where it grabs the sand grains and puts glue on them.
• After releasing the glue, it releases some proteins which contain a lot of DOPA - which crosslink the whole system and it hardens.

Question 37

How does complex coacervation work - probably the most complex bio-adhesion mechanism in marine systems?

Answer 37

Different glands or cell types. Some with acidic proteins and some with basic proteins. In the glands, you have the metals with the opposite charge which balances everything out. When the tube worm picks up a snad grain it releases all the componenets from the galnds into the building organ. The components dont set in the building organs as it is slightly acidic at around 6.8 or so and keeps all the components in a liquid form.

This electrically neurtral mixture of components is called a coacervate.

Incorperating water into the mixture gets around the problem of havin water at the interface.

Curing is stimulated by jump in pH from acidic (intracellular) to basic (seawater). This change in pH is enough for the transition from weak electrostatic interactions to ionic bonding.

Question 38

Similarities and differences of tubeworm and mussel adhesive.

Answer 38

Phragmatopoma californica adhesive has much in common with mussel adhesive, including polyanionic proteins created by phosphorylation of uncharged serinerich proteins. In mussels, Mfp-5 undergoes phosphorylation but serine levels are typically lower than in Phragmatopoma.

Coacervates of mussels and sandcastle worms are different, in that the mussel adhesive does not contain the preponderance of counter-ions found in the worm glue (and is, therefore, not necessarily electrostatically neutral).

Question 39

adhesive coacervates still have excellent physical properties for underwater adhesion:

Answer 39

However, adhesive coacervates still have excellent physical properties for underwater adhesion:

(1) They are denser than water and so can be directly applied to a surface without being diluted by diffusion and will sink;
2) They have low interfacial energies, enabling them to spread over wet surfaces;
(3) They have high internal diffusion coefficients, resulting in good mixing for cargo such as enzymes (e.g. catecholoxidase); - allows other compounds to pass through it.
(4) They possess shear- thinning properties. When you apply a force the whole thing will thin out, allowing it to be spread around on surfaces.

Question 40

Key point - whether the properties of a biofilm are close enough to that of an adhesive to be considered an adhesive themselves

Describe the composition and growth of a biofilm.

Answer 40

Basic scheme: (1) Immersion of a pristine surface – the ‘collector’;

(2) Adsorption, or the passive physical accumulation of a organisms on a collector surface (i.e. substrate);
(3) Attachment, adhesion, or the active consolidation of the interface between an organism and a collector, often involving the formation of polymer bridges between the organism and collector;
(4) Colonisation, or growth and division, of organisms on the collector’s surface;
(5) Planktonic dispersal.

Attachment of a cell to a substrate is termed ADHESION, and cell-to-cell attachment is termed COHESION….

Question 41

Describe the properties of a biofilm.

Answer 41

Once cells are adhered permanently to the substratum, they begin to divide and secrete large quantities of EPS. This is where a few scattered cells become a biofilm!

Biofilms, in general, display viscoelastic properties. In response to pressure, the biofilms go through a phase of elastic behaviour until a breakpoint is reached, after which the biofilm behaves like a viscous fluid

Sticky because they contain substances like alginate, xanthan and gellan gum aggregate in the matrix due to hydrogen bonding to form highly hydrated and stable viscoelastic gels.

Similarly, extracellular DNA (eDNA) is an integral part of the matrix, providing enhanced stability, separating it from other adhesives looked at.

Treatment of biofilms with nucleolytic enzymes (syn. nucleases) can result in deflocculation/disruption of the biofilm, whereas polysaccharide-degrading and protein degrading enzymes often have no effect.

Presence of nucleases can even prevent biofilm formation and interfere with adhesion to surfaces.

Metal ions such as calcium, iron and manganese, accumulate in and stabilise biofilms by bridging carboxyl groups across

Question 42

Composition of EPS in biofilm

Answer 42

• Polysaccharide, DNA and protein network creates a double or treble network system
  
  • Polysaccharides are major components of the EPS. Most are long molecules, linear or branched.
  • Proteins such as cell surface-associated and extracellular carbohydrate-binding proteins (called lectins), are involved in the formation and stabilization of the polysaccharide matrix and constitute a link between the bacterial surface and extracellular EPS…

Answer 43

• So, is biofilm a glue?

Adhesion mechanisms in nature are as diverse as the organisms that use them.

• However, there are many overlaps and similarities.
• What are the main convergent features and mechanisms?:
• Mechanical
• Physical
• Morphological
• Chemical and…
• Behavioural?
• Why do so many diverse organisms in different environments use similar mechanisms?

What are the advantages?

What are the limitations?

What are the key features?

• How are similar adhesive strategies optimised for different environments/applications?