Marine fouling and larval ecology

B subtilis polysaccharides

- "natto" is a Japanese delicacy: fermented soy beans, 1000 year old recipe - responsible for the "ropiness" in bread - can be used as a probiotic, put in animals feeds to prevent other more dangerous bacterium infecting the livestock - harmless, healthy factor in intestinal tract because it prevents other bacterium from settling) - industrial production of enzymes: amylase improves the conversion of starch to sugar so is important in the brewing process of alcohol

quorum sensing in V cholera ElTor

- 3 QS systems controlling: - expression of virulence at low cell densities - protease production, Hap protease (detachase) expressed at high cell densities - biofilm formation by upregulating genes for EPS (extra polymeric substances, slime) biosynthesis, expressed at low cell densities, makes colonies "sticky" - once colonies have infected host and become virulent, the switch off and exit host in order to spread further, expressing the "detachase" genes

CDP as antifouling paint system: mechanism and disadvantages

- CDP: controlled depletion paint - rosin: tree resin with biocide dispersed in the matrix, as matrix dissolved biocide is slowly released - problem with resin: it is resistant to degradation so as the biocide is released, there will just be a top layer depleted of biocide - disadvantages of CDP: - it is not self-smoothing, leaving a rough surface that increases drag - biocide release is not constant because of build up of leached layers - there is little activity during idle periods so hulls will foul when not underway - short lifetimes (3 years) and therefor higher costs because of applying new coats

D-amino acids

- D and L amino acids have exactly the same structure but are mirror images of each other - all biological life forms are made up of L form, all molecules in the body - when D forms occur, everything goes wrong - ie Thalidomide: anti-nausea and sedative drug for pregnant women to help morning sickness (also used in sleeping pills, 1950s), but gave birth to deformed children (no arms and legs, etc), the drug was banned - in the lab, only L forms occurred and was safe, but when manufactured using chemical synthetic processes it gave a mixture of L and D forms, and the presence of the D form caused defects - if D amino acids are added to biofilms, formation is dramatically disrupted - causes release of amyloid factors which results in the physical break up of biofilm

SPC as antifouling paint system: mechanism and advantages

- SPCs: self polishing copolymer - copolymer: made up of acrylic polymer +polymer attached to TBT, hydrophobic - the pigment dispersed in the coating is hydrophilic and lets water penetrate through this holes and reach the copolymer - as water cleaves the C-O bond between copolymer and the TBT, because alkaline seawater makes bond unstable, the toxin is released and the copolymer backbone is left with an unbonded Oxygen, leaving it to bond with free molecules and become hydrophilic and erodible -as it erodes, it leaves a smooth, not-yet-reacted layer of paint to release more toxins - constant renewal of the surface while the boat is underway, formulated to give a polishing rate of up to 5-20 um - because of the thin and stable leached layers, biocide release rate is continuous and constant, allowing for AF activity during stationary periods

natural mechanisms: models for antifouling methods

- Tursiops truncatus (bottlenose dolphin): has cutaneous ridges (250 um) running approximately normal to the flow, suggesting that dolphin skin is optimised for laminar flow (non turbulent), making it harder for organisms to attach - desquamation: skin flakes off and is completely replaced ever 2 hours, making sure ridges dont get full and smooth over, reducing vortices that form around the dolphin and reducing drag => could be used as a fouling release agent? - Globicephala melas (line-finned pilot whale): nanorough surface with nanoridges enclosing pores, gel coated skin containing enzymes involved in desquamation, lipidic area, hydrophobic and philic patches, air bubble cleaning when jumping - Steinberg and Kjelleberg group: used halogenated furanones of Delisea pulchra to prevent AHLs binding to their proteins, preventing quorum sensing at concentrations relevant to inhibition of colonisation

structure and differentiation in biofilms (3 mechanisms)

- Zobell (1943) discovered marine bacteria prefer to grow on surfaces - shear forces and hydrodynamic forces of flowing water affect structure - complex architecture within colonies - 3 mechanisms lead to biofilm formation, which can all happen at the same time and may take up to 10 days - redistribution of attached cells by surface mobility (ie type IV pili mediated twitching in P. aeruginosa) - binary division of attached cells - planktonic recruitment

direct development

- absence of a free-living stage, hatch as juveniles - for protection, often seen in eggs strings (cephalopods) - development takes place within egg capsules and the juveniles are fully developed because hatchng

convergent evolution

- acquisition of the same biological trait in unrelated lineages - arrises due to restrictions on the number of potential solutions that can successfully evolve in response to a similar survival pressure - may look superficially similar because they perform the same function (shapes of sharks and dolphins or mammal eyes and cephalopod eyes) - examples in adhesion mechanisms are widespread, suggesting that there are severe limitations on the number of ways organisms can stick to surfaces

Dispersin B

- an enzyme produced by the Gram negative oral pathogen Actinobacillus actinomycetemcomitans - the pathogen sticks tenaciously to glass, and the enzyme hydrolyses the glycosidic linkages of PNAG (the stuff that makes it sticky)

reasons for biofilm dispersal

- antagonism: being attacked, lack of room - dispersal is a natural part of life - fresh nutrients - new space for growth

desperate larva hypothesis

- as larvae age, they are more likely to settle on unfavourable surfaces - their lipid stores are depleted as they age - when they are young, they are choosy and most dont settle, choosiness decreases with age - ie proteins of adults stimulates cyprids to settle (barnacles are gregarious as cross fertilization is obligatory and proximity to potential mate is required) - applies in particular to larvae that dont feed in the plankton - each phase of settlement is evoked by a specific releaser/inducer (polar, primary metabolites with the exception of non polar AHLs, that the organisms should be preadapted to using)

biofilm development stages

- attachment - microcolony - subpopulation interactions (quorum sensing, communication) - macrocolony - activation - dispersion

Vibrio cholera

- bacteria that float around in non-virulent form, then release a dangerous toxin that causes a serious bacterial infection of the small intestine, breaking down the lining and causing explosive diarrhea - if untreated 60% die, kills 10,000 people per year - the bacteria is adapted to survive in the sea and in the human gut (very successful because it can adapt) - when it reaches the gut, it forms a biofilm and this causes them to be dangerous as they produce potent enterotoxin - attachment to surfaces and biofilm formation is central to ability to cause disease

diffusible signals: quorum (density dependent) sensing

- bacteria use acylated homoserine lactones (AHLs), also known as auto-inducers, to signal critical population densities: known as quorum sensing - links the expression of genes to a critical population density - involved in regulation of many phenotypes - bioluminescence - swarming - biofilm differentiation - conjugation - antibiotic biosynthesis - pathogenesis - these signals may have resulted in the evolution by higher organisms to interfere with, escape from, or exploit signalling pathways to their advantage (Lyon and Muir 2003)

biofilm maturation

- biofilms thicken and change in properties, forming a complex 3D structure - channels and pores appear - bacteria are redistributed away from the substratum - activation: a stage of differentiation, reverse of planktonic to sessile transition, cells produce and release enzymes to chop up the biofilm, grow flagella to become motile - seek out uncolonized areas, disperse - QS mediated AHLs are required for mature biofilm formation - gene expression varies depending on where the cells are within the biofilm

what are the main organism groups that foul

- biofilms: microbial biofouling (exopolymeric substance: brown slime) - bacteria, cyanobacteria, unicellular algae, protozoa, fungi, diatoms (dominate) - macrofouling: macroalgae - seaweeds - invertebrates - sponges, cnidaria (hydroids, anemones), bryozoans, annelids (tube-forming polychaetes), molluscs (limpets, mussels), crustaceans (barnacles) - urochordates - tunicates (sea squirts either colonial or solitary)

broadcasting vs brooding

- body size has a bit influence on reproductive mode - ie: mathasterias glacialis and asterias rubens are broadcast spawners because of large body size, can produce may eggs, planktotrophic larva - bloody henry star (henricia oculata) is a brooder with lecithotrophic direct development because it is small and cant produce many eggs, so produces few and protects them with body (stands on tips of arms and broods under her)

what do future antifouling agents need to be to be effective?

- broad spectrum without biocides: effective against all different phyla instead of one target species - durable enough for navy ships (drivers of the industry): 12-year docking cycle with limited or no hull cleaning - a greater understanding of biological fouling process is necessary!

biofilm disruption mechanisms of bacteria

- c-di-GMP inhibits nucleotide biosynthesis and DNA replication, shows antibiofilm activity - nitric oxide releases biofilms from surface - alginate lyase in P aeruginosa chops up polysaccharides - DSF (diffusible signal factor) released in Xanthomonas campestris, a plant pathogen, that induces a release of manA encoding a beta-mannanase (which breaks down sugars that holds biofilm together)

larval senescence

- can a larva remain a larva forever or does it grow old? - ability to metamorphose, adult performance and fitness decreases

case study: suction in cephalopods

- cephalopod sp (Nautilus sp, sepia sp, euprymna sp, idiosepius sp) attach to surfaces by suction and by liquid adhesives - some also use a de-adhesive (euprymna sp) that digests glue and releases prey - use of liquid glue in cephalopods is an ancestral trait, more advanced cephalopods rely more on suction - octopuses are the only animals that use suction as only form of adhesion - use hundreds of thousands of suction cups to achieve strength - other animals compound effect with little teeth or "glues" - ie squid cuttlefish use liquid glue and little teeth on suction cups

ROI and RNI as signals

- chemicals are very damaging so it is essential to detect and act upon these signals - many signalling pathways in both prokaryotic and eukaryotic systems - detection of the chemicals lead to stress responses - role of RNI and ROI in biofilms unknown - some stress responses include dispersal (ie P aeruginosa biofilms exposed to NO caused dispersal in the nanomolar range (experiment by Barraud et al))

brief larval duration in ascidians

- colonial ascidians (ie Botryllus schlosseri) increase the size of their colonies by budding to produce more zooids - those that undergo sexual reproduction produce tadpole larvae (with nerve cord and notochord) with a swimming life of a few hours to move a short distance from the parent colony

dispersal in P aeruginosa

- colony is formed and the core of the colony is later evacuated leaving behind a hollow shell-like structure - when nutrient levels reduce, cells form cooperative structures with reduced metabolism, involving quorum sensing and differentiation - leads to lysis of a subpopulation of cells within the microcolony and the generation of oxidative stress - reactive oxygen intermediates (ROI) - superoxide (O2-) - Hydrogen peroxide (H2O2) - Hydroxyl radical (HO.) extremely reactive

duo-gland adhesive delivery

- common in organisms that use curing adhesives, reason unknown - DOPA (present in adhesive of mussels) and PS (Phosphorylated serine) are also found in organisms from different phyla

mussel adhesion: structure of a byssal thread

- composed of collagen and several unique proteins - top section: proximal byssus, a bundle of collagenous fibers (Col-P) in a protein matrix - middle section: distal byssus, uniform alignment of Col-D fibres at its core surrounded by jumbled fibres and a varnish outer coating - bottom section: a thin layer of adhesive (Mefp-5) underneath a disc of foamy material into which the thread is embedded

timing/location of fouling

- conditioning layer apparent within seconds of immersion (proteins, dna, general chemicals from the water), which prepares the surface for subsequent colonization - seaweeds act as pioneers (as they grow and reproduce quickly enough not to be overgrown), changing the surface making it easier for other organisms to attach - important fouling species are those with short generation times - fouling generally happens with in port or harbor rather than underway (lots of artificial surface for potential foulers so large populations found in harbors, protection from predators, large food supply due to proximity to urban development) - limiting factor is space not food or potential colonizers

barnacle adhesion: protein structure

- cured proteins are amyloids - fibrillar nature of the proteins prevents crack-propagation - 100 kDa and 52 kDa proteins are hydrophobic and highly insoluble: self assemble into a rigid framework - 68 kDa and 19kDa are soluble and function in a non-covalently bonded state: prime the surface, set into the framework and harden by disulphide bonding

adhesion in barnacles: process

- cyprid cement glands contain two types of cells (dual gland system) that harden when mixed - to keep them separate, precursors stores in granules in alpha and beta cells, which undergo exocytosis when stimulated with dopamine = mix of granules - mixture travels down the antennules from the glands, embeds the antennules in a blob of glue on the surface which hardens and sticks there - polyphenoloxidase connects proteins together, natural polymerizer = quinone tanning? - sometimes proteins are released first to prep the surface and displace water before cement is secreted - after metamorphosis, adult barnacles produce a different, stronger, adhesive; not calcium carbonate! it is not just an extension of the shell

planktonic to biofilm transition

- deregulation of many gene systems such as the flagellar apparatus since movement is no longer required - upregulation of gene systems such as a carbohydrate metabolism: secrete polysaccharides that make up bacterial slime layer - expression of biosynthetic pathways for secondary metabolites (organic compounds not involved in growth, reproduction, or development): production of antibiotics

complex coacervation

- different glands with different contents - in the granules: proteins with acidic peptides, oppositely charged polyelectrolyes, and lots of divalent cations - after exocytosis, these mix to form an electrostatically cohesive fluid coacervate, a fully hydrated open network - after adhesion, curing is stimulated by a jump in pH from acidic (intracellular) to basic (seawater) - ionic bonding between Ca2+ and phosphoserine

DSF in biofilm formation/regulation

- diffusible signal factor - especially used in genus Xanthomonas - small fatty acid - organic communication molecule - upregulates biofilm formation

intermediate larval duration

- duration measured in days, is very common in many pelagic lecithotrophic marin inverts - gastropod molluscs and polychaete annelids - sometimes pick up cues for good settlement sites from other animals (ie nudibranchs from bryozoans)

Drivers for new/improved coatings development

- economic considerations: operational costs like bunker fuel and the cost of coatings that keep the ship clean - improved performance: companies are trying to get a competitive edge and maintain their reputation - legislative pressure: from environmental lobbyists about biocide use to consider all consequences for human and environmental health - reduced emissions/translocation of invasive species

rhl quorum sensing system in P aeruginosa

- example of multiple quorum sensing systems - intricate complex regulatory connections - rhlr: regulatory protein - rhlI: inducer protein - not independent control system, but works as part of a cascade with others including LasR and LasI - under anaerobic conditions, cells which lack rhl circuit commit metabolic suicide due to NO intoxication (NO is one of the few gaseous signalling molecules known, it is a free radical and highly reactive) - Las and rhl quorum sensing systems function via signals N-(3-oxododecanoyl) HSL (3-oxo-C12-HSL) and N-butyryl HSL (C4-HSL) respectively - rhlA (positively controlled by rhl quorum sensing system) is required for production of rhamnolipids, surfactants important for virulence - in this way, expressions of many genes in response to environment can be finely tuned

sources of chemical cues

- habitat-specific chemical cues are derived from conspecifics, host organisms, prey, or biofilms

antagonistic disruption of biofilms case study and experiment

- hypothesis: aquatic bacteria disrupt the biofilms of their competitors during growth on surfaces - ability to dissolve slime layer of other bacteria - quantification of biofilm dispersal - dip slides into solution of crystal violet dye which dyes the slime layer blue/violet - dip slide into solution of other bacteria - biofilm with color disappeared - bacteria had dissolved their biofilm (using BDC: biofilm dispersing compound) and escaped into solution after being attacked - ie Micrococcus luteus strain LY appears to be a potent BDC producer with activity against other and its own biofilms - reduction in biofilms exposed to BDCs produced by marine isolates

confocal microscopy

- individual cells can be tracked over time - confocal laser scanning microscope zooms in on a focal plane of a 3d structure - showed that - surface associated lag for primary biofilm cells as they adapt to sessile environment or undergo a phenotypic change - secondary biofilm cells are already adapted to life in a biofilm so grow faster

BDC: biofilm dispersal compound

- induces physiological response in target strains growing in biofilm - bacteria respond by dissolving their biofilm and swimming away, starting a planktonic lifestyle - the bacteria stay alive as there isnt an antimicrobial involved - other agents are needed to kill them

fouling release as an antifouling method

- interferes with the adhesion of organisms - for this method to work, the anti fouling efficacy needs to be increased - right now, they do not prevent fouling, but make it easier to detach the organisms, but in the future they need to stop adhesion in the first place

relationship between larval duration and adult habitat type

- intertidal soft sediment mostly species that had non-planktonic larvae, little that lives on the sediment but mostly burrows into it, incoming tide is sufficinet to move them around to colonise near areas - subtidal soft sediment have long lived planktotrophic larvae, as species have less risk of being pelagic and finding nowhere to settle - intertidal rocky shores had highly variable larval duration and developmental modes, but mostly planktonic larvae, characterized by grazing molluscs, polychaete worms, bryozoans - fine sediment far shore mostly long lived planktonic for dispersal or nonplanktonic for colonial species

examples of booster biocides

- irgarol 1051: interferes with photosynthesis of algae, but has little activity against animals - diuron: urea based herbicide, inhibits photosynthesis - sea-nine 211: isothiazolone active against soft fouling - zinc and copper pyrithiones: bactericides/ fungicides, active against soft fouling - EU biocidal products directive: a number of biocides will no longer be available for use

furanones

- isolated from D pulchra (seaweed species), which releases compounds that inhibit quorum sensing mediated biofilm formation - compound identified as halogenated furanones (Bromine) - similar in structure to AHLs and block AHL mediated communication

Bacillus licheniformis EI-34-6

- isolated from seaweed Palmaria palmata - produces red pigment and bacitracin when growing in reactor that mimics seaweed surface, the air membrane surface (AMS) bioreactor - if growing in AMS reactor it grows in a biofilm

rhamnolipids

- key role in maintaining the 3D architecture of biofilms (channels and pores) - affect epithelial cells in human respiratory tract - airway mucosa protects against infection as it guards receptors using mucus to prevent pathogen binding - P aeruginosa cant penetrate healthy mucosa but can do in immunocompromised or cystic fibrosis patients - rhamnolipids help break down mucosal barrier

non-pelagic lecithotrophy

- large eggs - males shed sperm onto surface, females irrigate the burrow wit the sperm to fertilize, females will stop feeding so as not to ingest the egg - larvae remain closely associated with the surface sediment in mucus tubes - larval life 2 weeks - juveniles surf up the beach on incoming tide to move further up to "nursery" where they grow to avoid competition

pelagic lecithotrophy: characteristics

- large eggs that contain all nutrition required to carry the larvae through to juvenile stage without feeding, carry 25x as much yolk as a planktotrophic egg - short larval life (days) - lack specialized feeding appendages despite having a developed gut, but develop structures to use immediately after settling out

planktotrophy: characteristics

- larvae derived from small eggs (70-80 um, nucleus occupies 10% of oocyte, remainder is lipoprotein yolk and lipid stores) - long larval life - specialized mechanisms or apparatus (ie starfish bipinnaria has large ciliated oral field with cilia to collect food)

larval longevity

- larval duration can be anything from minutes to years - species with non-feeding larvae have finite resources and limited life span, need to find suitable location before resources run out - feeding larvae have the possibility of delaying metamorphosis to the benthonic adult until a suitable settlement site is located - site settlement depends on space availability at the time, which species are spawning (which larvae are available)

capillary adhesion

- liquid "prefers" to stick to the glass rather than to itself, so it is drawn up a glass tube - glass has a lot of "free energy": capability to form new chemical bonds, since it has more than liquid does, liquid prefers to stick to glass

properties a coating should possess to resist adhesion

- low elastic modulus (energy used to stretch the surface) - high molecular mobility in the backbone and surface-active side chains - a thickness that can control mechanics of the interface, the thicker the coating the easier it is to release organisms such as barnacles - smooth surface to avoid infiltration of adhesive, prevent "lock on" effect of some adhesion - lubricity

biocidal antifouling agents

- mainstay for the foreseeable future - most coatings have toxic ingredients - industry is trying to find ones with improved environmental profile - less compounds released, with shorter half lives - compounds that are easily broken down, are not persistent - goal is to move away from biocides, problem is they are the most effective

mussel adhesion: mechanism

- produce a byssus of elastic adhesive threads designed to re-distribute the load and dissipate energy - mesh of threads so that if attacked, force is distributed across lots of threads - threads are cast by glandular tissue in the foot of the mussel out into a groove

case study: adhesion in arachnids

- many arachnids, and insects, use hair pads rather than smooth, flexible ones - the hairs provide many individual contacts each requiring energy to remove from the surface = increased difficulty to remove - prevents crack propagation: if one section "detaches" or slips, the others will still hold - since most insects are not large enough to use boundary layer friction on their contact points, they use non-newtonian fluids: microscopic droplets of water in the oil form an emulsion that stiffens under strain; the more water the more resistance to shear

chemical cues (settlement) from biofilms

- many organisms respond to biofilm at settlement - ie hydroides elegans delays settlement until encounters biofilm - magnitude of induction correlates with bacterial density - the specific cue is unknown, only suggested that it is a surface cue - in order to form the biofilm in the first place, communication is involved as cells need close proximity to exchange molecular signals that regulate behaviour

Thorson's rule

- marine benthic invertebrates at low latitudes have small eggs that give rise to planktotrophic larvae, whereas those at high latitudes have large yolky eggs or large eggs with brooding

prolonged larval duration

- measured in weeks or months, common in pelagic planktotrophic inverts (ie sea urchin echinopluteus, star fish brachiolaria) - spiny lobster phyllosoma have larval duration of years - settlement site is not critical as long as its close to a food source - larvae is symmetrical, so needs to become radially symmetrical (takes longer): the juvenile starts growing on the larva and then larval structures are reabsorbed

case study: suction in limpets

- modern day limpets use suction for adhesion for attachment during locomotion as well as capillary/stefan adhesion - when exposed by the tide, limpets secrete a true adhesive hydrogel, containing <97% seawater, several polar proteins and a 140 kDa glycoprotein complex for adhesion - forms a scaffold that hold water together forms a sticky mucus

lock and key (curing) adhesives

- most commercial adhesives work via chemical reactions with the surface, followed by mechanical interlocking of the hardened adhesive with micro-scale surface features - cyanoacrylate: reaction is catalysed by the presence of water (OH- ions specifically) and is an example of anionic polymerisation

biological adhesives

- natural polymers (compound with repeating units), proteinaceous - some polymers show up in many organisms' adhesives

importance of adhesion

- on land adhesion would be a bad idea because predation cant be escaped and dispersal cant happen - but in marine environments, the ocean currents do most of the work: brings food, disperses gametes, takes away waste - it is energetically favourable - once decision is made to become sessile, it needs to be done properly because dislodgement could be fatal - it is hard to find surfaces now that organisms are not adapted to stick to => fouling is a problem!

Pseudomonas aeruginosa

- opportunistic human pathogen - gram negative, aerobic although can grow in microaerobic habitats - infects lungs (common in patients with cystic fibrosis, as they produce thicker mucus around the airways, ideal habitat for bacterial infection) and also produces alginate biofilms which reduce oxygen transfer - fluid genome means it can adapt to new environments and allows transfers of mutations to offspring very quickly

lag phase in the planktonic to sessile transition

- organisms that initially colonize a "clean" surface are referred to as primary biofilm cells, descendants are secondary biofilm cells - Planktonic, primary, and secondary biofilm cells of Pseudomondas aeruginosa PA01, which produce GFP, are used to study transitions (proteins can be tracked carefully and visualised, growth of individual cells can be monitored, bacterial cells can be labelled) - experiments performed in a parallel plate flow cell reactor with a glass substratum

tributyltin compounds: effect

- organotins= organic molecule +tin atom - was the most efficient anti-fouling compound used to date - caused the crash of the Arcachon Bay Oyster industry 1970s and imposex in dogwhelks - Legislation to control use of TBT anti-foulants: 1982 France banned use on small crafts, other countries followed, 2003 ban on larger vessels, 2008 total ban - alternatives are either toxic (copper, which is not broad spectrum, and booster biocides) or fouling release, which are both more expensive and use of copper on aluminium boats risks galvanic corrosion

ambulatory organs of cyprids

- paired antennules made up of segments, organ of attachment (hairy attachment disc for adhesion) and sensory armament (mechanosensory and chemosensory chaetae)

mussel adhesion: process

- peptides released by the glands contain large quantities of phosphorylated serine residues and L-DOPA - redox-active catechol side-chains on L-DOPA crosslink under catalysis by polyphenoloxidase - outcompete water for hydrophilic surfaces because of their surfactant-like properties, which contain high% of water themselves and are basically hyrdogels

how is marine biofouling controlled

- physical scraping and mechanical removal (dry docking, divers) - toxic techniques - lead sheathing - copper based paints - tributyltin compounds - non toxic future techniques? - back to copper based coatings? - silicone bases non stick paints - attack the fouling organisms at their vulnerable point

options for development for broadcast spawners

- planktotrophic: planktonic and feeds whilst a larva, long larval life - pelagic lecithotrophic: planktonic but does not feed, has short pelagic life - non pelagic lecithotrophic: remains closely associated with the benthos during larval life - direct: no free-living larva and emerges from the egg as a fully formed juvenile

Bacillus subtilis

- preferred Bacillus model bacteria used in studies because it cant cause disease and is easy to genetically manipulate - gram positive, can explore the same genetics of g positive cells using this - common soil organism although found in many environments - spore forming cells, for dispersal and survival of adverse conditions

Nitrosative stress

- reactive nitrogen intermediates (RNI) - Nitric oxide (NO) - Peroxynitrite (ONOO-) - Nitrous acid (HNO2) - Nitrogen trioxide (N2O3) - RNIs are produced continuously during anaerobic respiratory metabolism, if there are too many DNA damage and death occur - Nitric oxide intoxication leads to cell death - cells have to develop protection to protect against oxygen and nitrogen

why is marine biofouling a problem (5)

- reduces speed and maneuverability of ships - greater fuel and maintenance cost (total saved with effective AF/FR coatings $3B p.a) - increased environmental impact through increased fuel use/emissions (90,000 vessels emitting same CO2 as 190million vehicles, increased drag is bad) - problems for static structures - destabilizes through increased loading - enhances the corrosive process - fouling of intake and outflow pipes for coastal power plants - problems for the aquaculture industry - reduced water circulation - vectors for disease - invasive species

regulation of epsA-O and tapA operons in Bacillus biofilms

- regulated by master switch called Spo0A, a transcription factor - operon: by putting all genes together that code for one gene, the cell is more efficient at switching that gene on and off - effect of Spo0A will depend on its concentration - at low levels, it upregulates the operons and 3D colonies of biofilm are created - at high levels, they are switched off, biofilm is not created, and the sporulation genes are switched on - matrix producing cells become cannibalistic, using dead cells as nutrition - cells making biofilm produce toxins: SDP (sporulation delaying protein) and SKF (sporulation killing factor) - also produce immunity proteins to defend agains the toxins so that the toxins only kill bacteria and other cells not expressing immunity to regulate whats best for particular portions of the biofilm

adaptations for long pelagic life

- remaining suspended in water column - small larvae (trochophores, veligers) use cilia - larger larvae (zoea, brachiolaria) use appendages "arms" or presence of spines - feeding (ie cilia) - ability to delay metamorphosis (which may decrease chances of success, growth, fecundity, and overall fitness)

non-attractive adhesion: suction

- requires an elastic cup which is initially contracted and then expanded to form a zone of reduced pressure - creates a vacuum, no pressure can be pushing outside the suction cup, only onto it - can never exceed adhesion of 1 atmosphere

settlement inducer of Holopneustes purpurescens

- sea urchin that lives in canopy of algae, Delisea pulchra and Ecklonia radiata - settlement induced by water soluble histamine produced by D. pulchra (4.5 um)

case study: adhesion in tree frogs

- secrete a liquid adhesive from their soft toe-pads, producing a capillary effect that sticks to the leaves - capillary adhesion is weak in "lateral shear", so frogs also use a boundary-layer friction between their cuticle and the surface to prevent slip: grooves on the toe pads and release a sticky mucus

succession hypothesis (and examples)

- sequence of events following immersion of surface is predictable - preventing macrofouling by controlling microbial fouling - ie hydroides elegans needs biofilm in order to settle, larvae can maintain competence for several weeks, dramatic metamorphosis within minutes of exposure, dont settle next to their own kind, colonize clean surfaces - ie ulva spores do not need a biofilm to settle but the more there is, the faster they settle, mostly near the waterline where there is the most light, some strains of bacteria cause settlement (either on microcolonies or randomly), some inhibit - ie balanus amphitrite cyprid settlement is inhibited by natural biofilm, older the biofilm, the more it inhibited, inhibition by bacteria is density dependent - ie hydroides dianthus settle gregariously, induced by biofilm and conspecifics, as larva age they are more likely to settle even without conspecifics

enzymes that interfere with adhesion

- serine proteases have been found to interfere with adhesion of barnacles, algae, and bacteria

navy as the driver for antifouling

- spends more than half of its time in port (55% for aircraft carriers for three months at a time, 56% for cruisers/destroyers, for 2 days at a time) - a commercial tanker spends 85% of time underway

surface tension and surface energy

- tension: forces of attraction between the molecules that make up the liquid, the force that holds a liquid together - energy: excess energy associated with the presence of a surface, can change the shape of a water droplet

larval competence

- the acquisition of the ability to undergo matamorphosis to the benthonic juvenile form, often in response to a prey derived or conspecific derived chemical cue (metamorphic inducer) - at some stage a larva reaches a stage when it is competent to metamorphose, which may involve changes to larval structure from feeding form to settling form (ie loss of ciliated velum in mollusc veliger larvae) - metamorphose in response to a chemical cue (ie barnacle cue from adults, prey cue from bryozoans to nudibranchs), can only be induced in a physiologically ready larva

define adhesion

- the action or process of adhering to a surface or object - different scales of adhesion have different mechanisms

case study: adhesion in bush cricket

- the tarsi have smooth flexible pads that are used for surface attachment, design very similar to the tree frog - also release a cuticular oil from between the lamellae of these pads, mediating adhesion in a similar way to tree frogs

chemical basis of barnacle gregariousness

- three pheromones recognized - adult glycoprotein: the settlement inducing protein complex (SIPC), water soluble cuticular protein (binds to all parts of the body lined by cuticles), physiochemical characteristics similar to insect arthropodin (Crisp) - waterborne cue: Rittschof experiment found that water that had been conditioned by adult barnacles could induce larvae to settle, as long as the petides had neutral or basic carboxyl terminal they could be mimicked (like GGR). Newcastle findings: small molecule, <500 Da, induced searching behaviour - temporary adhesive serves as settlement pheromone, is proteinaceous, leaves "footprints" that stimulate conspecific settlement, related to SIPC

examples of medical applications for antifouling mechanisms

- toothpaste/mouthwash to clean plaque off teeth - contact lens cleaning solution - artificial knee surgery: if infection occurs as a result of bacteria the only treatment is to remove the artificial joint and replace, which is traumatic for the patient, and uses expensive resources, so applying marine enzymes to break down biofilms in medical conditions is huge! - surface of artificial joints now have nanotexture that is more ridgey and bumpy because microbes prefer not to settle onto surface with texture

effects of latitude on larval developmental mode

- towards the poles it is advantageous to have lecithotrophic larvae - cold temperature with less seasonal variation (dev takes longer at cooler temps), less sunlight, no extended blooms but very short bloom of food (needs exceed the availability of food) - intermediate latitudes have strong seasonal signals that influence nutrient availability (more in spring but not enough light for phytoplankton to bloom), two blooms a year

Characklis' classification of biofilm development

- transport of org molecules and bacteria towards a submerged surface - adsorption of molecules (conditioning layer) - attachment of bacteria to conditioned surface - metabolism of organisms: faster adherence - growth - detachment

Bacillus biofilms

- two main components: - exopolysaccharide (epsA-O operon) - Amyloid fibres (tapA operon) that hold biofilm together, proteins - these are long polymers that have sticky properties, well adapted for providing protective layer of slime to coat the cells - heterogenous: within the biofilm several cell types exist with different jobs - motile cells - matrix producing cells - dormant spores

why are marine chemical cues difficult to characterise?

- unstable - tightly complexed with other molecules so difficult to get at - present only in trace amounts, lower than detection limits of instruments - rapidly diluted (in case of waterborne) - availability of reliable assays - lack of knowledge of how to culture larva

define marine biofouling

- unwanted fouling of man-made marine structures by marine micro organisms, seaweeds, and animals - it is NOT colonisation or growth on natural hard surfaces or living surfaces

adhesion in barnacles: mechanism

- use hairy attachment pads and use a liquid secretion - leave behind a "footprint" - density of hairs made out of fibrillar material: works like velcro in that many little strands need to break and lots of energy needs to be put in to detach - nature of the liquid is proteinaceous - adhesive proteins have sacrificial bonds: - tertiary structure of proteins and between proteins that dissipate energy - each bond needs to break, absorbing the energy that is put into it, before detaching - prevents crucial backbone from rupturing - when temporary adhesion is no longer enough, a permanent solution is needed

defining reproductive modes in larval ecology (4)

- viviparity: release of fully formed juveniles (rare in marine invertebrates) - brooding with parental care (some cephalopod molluscs): large female investment, energy needed to irrigate the eggs until they hatch, only reproduce once - egg laying without parental care (gastropod molluscs) - broadcast spawning (most common with marine inverts and especially fouling organisms): eggs and sperm released, fertilized, produce a larvae, variable larval duration, settles and forms parental population/colony

contact angles on a surface

- when a liquid does not spread on a substrate, a contact angle q is formed - balance between the forces on the surface is give by Young's equation: g(s)=g(sl)+g(L)cosq where g(s)= surface tension of solid surface, g(l)= surface tension of the liquid, and g(sl)= boundary tension between the solid and the liquid - low energy, low angle - ie, the lotus leaf ia a low energy surface hydrophobic, water stays in droplets on the surface of the leaf

spore conversion in Bacillus subtilis

- when survival conditions are bad, bacterial cell concerts itself into a spore (sporulation) - regulated by 40-50 genes, which tell the cells to produce hard coat, package DNA, and break open mother cell (which will decay) and release spore) - packages DNA into a hard seed/pore which is resistant to harsh conditions (temp, UV) and can survive for hundreds of years - can be dispersed by wind or water - when spore finds suitable conditions for growth (moisture, nutrients, warmth), it will germinate and bacterial colony can grow again

stages of cyprid settlement behaviour

- wide exploration (stick reversibly to take steps over substratum, temporary attachment by alternate attachment and release of adhesive tips of the antennae (Visscher 1928)) - close exploration (when they sense or sample a favorable environment, take frequent turns in order to remain in general vicinity (Crisp 1984)) - inspection (zone in on one site, commit to permanent attachment)

advantages and disadvantages of fouling release agents

- work well on fast moving vessels >20 knots (but not many ships go this fast) but have a persistence of slime >30 knots - easily damaged - difficult to clean underwater - expensive - do not prevent fouling (slime is a major issue), act mainly on adhesion of adult/mature organisms - SOLUTION: current research aimed at increasing effectiveness of silicones to be more robust and self clean at lower speeds

non-attractive adhesion: stefan adhesion

- works by resistance to viscous flow