Tuning orb spider glycoprotein glue performance to habitat ... · Spider Catalog, 2017). The order Araneae is composed of two suborders: Mesothelae, which have segmented abdomens

REVIEW

Tuning orb spider glycoprotein glue performance to habitathumidityBrent D Opell1 Dharamdeep Jain2 Ali Dhinojwala2 and Todd A Blackledge3

ABSTRACTOrb-weaving spiders use adhesive threads to delay the escape ofinsects from their webs until the spiders can locate and subdue theinsects These viscous threads are spun as paired flagelliform axialfibers coated by a cylinder of solution derived from the aggregateglands As lowmolecular mass compounds (LMMCs) in the aggregatesolution attract atmospheric moisture the enlarging cylinder becomesunstable and divides into droplets Within each droplet an adhesiveglycoprotein core condenses The plasticity and axial line extensibilityof the glycoproteins are maintained by hygroscopic LMMCs Thesecompounds cause droplet volume to track changes in humidity andglycoprotein viscosity to vary approximately 1000-fold over the courseof a day Natural selection has tuned the performance of glycoproteincores to the humidity of a speciesrsquo foraging environment by altering thecomposition of its LMMCs Thus species from low-humidity habitshave more hygroscopic threads than those from humid forestsHowever at their respective foraging humidities these speciesrsquoglycoproteins have remarkably similar viscosities ensuring optimaldroplet adhesion by balancing glycoprotein adhesion and cohesionOptimal viscosity is also essential for integrating the adhesion force ofmultiple droplets As force is transferred to a threadrsquos support lineextending droplets draw it into a parabolic configuration implementinga suspension bridge mechanism that sums the adhesive forcegenerated over the thread span Thus viscous capture threadsextend an orb spiderrsquos phenotype as a highly integrated complex oflarge proteins and small molecules that function as a self-assemblinghighly tuned environmentally responsive adhesive biomaterialUnderstanding the synergistic role of chemistry and design in spideradhesives particularly the ability to stick in wet conditions providesinsight in designing synthetic adhesives for biomedical applications

KEY WORDS Adhesive Biomaterial Hygroscopic Prey captureSelf-assembling

Introduction ndash spider diversity and the role of prey capturethreadEvolution in silk use has played a crucial role in the success of thediverse over 47000-species-strong arachnid order Araneae to whichspiders belong (Vollrath 2005 Vollrath and Selden 2007 WorldSpider Catalog 2017) The order Araneae is composed of twosuborders Mesothelae which have segmented abdomens likescorpions and spinnerets that extend from the middle of their

abdomenrsquos ventral surface and Opisthothelae which haveunsegmented abdomens and posterior spinnerets (Platnick andGertsch 1976) Opisthothelae contains two infraordersMygalomorphae which includes tarantula and trapdoor spiderswhose cheliceral fangsmove parallel to the bodyrsquos sagittal plane andAraneomorphae which contains over 95 of all living spiderspecies whose fangsmovemore perpendicularly to the sagittal planeAraneomorphae origin coincided with the appearance of a cribelluma spinning plate formed of thousands of spigots that produces thenanofibers of a dry fuzzy capture thread termed cribellate thread (seeGlossary) Although some araneomorphs continue to spin cribellatethreads (Opell 2013) most no longer do so constructing webs thatare not sticky or like jumping spiders and wolf spiders abandoningweb use in favor of other hunting tactics The first orbwebs containedcribellate threads but 110 million years ago members of thesuperfamily Araneoidea replaced these with moist viscous capturethreads (see Glossary) (Pentildealver et al 2006) These viscous threadsare considered a key innovation (Bond and Opell 1998)contributing to the diversity of this clade which contains 26 ofall spider species and comprises 17 families of orb-weaving spidersand their descendants that spin webs with divergent architectures(Blackledge et al 2009ab Dimitrov et al 2016 Hormiga andGriswold 2014)

Organisms employ adhesive secretions for a variety of otherfunctions For example Polychaeta annelids construct protectivetubes from cemented sand particles (Pavlovic et al 2014) barnaclescement their cases to rocks and mussels attach themselves by byssalthreads to the substrate to avoid being swept away by currents(Kamino 2010 Waite 2017) Like most commercial adhesivesbioadhesives typically have an initial low-viscosity phase duringwhich they establish surface contact followed by a phase ofincreased stiffness which allows them to resist the crackpropagation that leads to failure (Gent 1996) English ivy clingsto tree trunks by secreting a low-viscosity adhesive solution thatspreads before water evaporates hardening it into a matrix (Huanget al 2016) However the challenge is much greater for aquaticanimals (Stewart et al 2011) Barnacles and mussels solve theproblem by secreting adhesives that are subsequently enzymaticallyhardened (Dickinson et al 2009 Naldrett 1993 So et al 2016Waite 2017) By contrast the glycoprotein (see Glossary) glue ofan orb-weaving spiderrsquos viscous threads remains hydrated andpliable in air because it is contained in tiny aquatic spheres (Fig 1DE)(Edmonds and Vollrath 1992 Tillinghast et al 1993 Townleyet al 1991) This ensures that their glycoprotein adhesive retains itsviscoelasticity for effective adhesion (Sahni et al 2010)

Orb-weaving spiders integrate silk produced from four distinctsilk glands into a highly effective prey capture web (Fig 1A)Attached by pyriform gland (see Glossary) secretions (Sahni et al2012a Wolff et al 2015) non-adhesive radial and frame threadsproduced by major ampullate glands (see Glossary) absorb anddissipate the kinetic energy of an insectrsquos impact (Sensenig et al

1Department of Biological Sciences Virginia Tech Blacksburg VA 24061 USA2Department of Polymer Science Integrated Bioscience Program The University ofAkron Akron OH 44325 USA 3Department of Biology Integrated BioscienceProgram The University of Akron Akron OH 44325 USA

Author for correspondence (bopellvtedu)

BDO 0000-0002-1830-0752 AD 0000-0002-3935-7467 TAB 0000-0002-8166-5981

1

copy 2018 Published by The Company of Biologists Ltd | Journal of Experimental Biology (2018) 221 jeb161539 doi101242jeb161539

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2012) while spirally arrayed adhesive prey capture threadsproduced from flagelliform and aggregate glands (see Glossary)retain the insect (Sahni et al 2013) until the spider can locate run toand begin to subdue it Material invested in non-adhesive threadsinfluences the size and velocity of insects that a web can stop(Sensenig et al 2012) and material invested in capture threadaffects the time an insect is trapped (Opell et al 2017) A retentiontime (see Glossary) difference of even a few seconds can be thedifference between a prey being captured or lost (Eberhard 1989)

Orb-weaving spiders are not unique in relying on extendedphenotypes (see Glossary) for important functions (Dawkins1982) nor are they the only animals that use these products forprey capture For example parchment worms and caddisfly larvaeemploy nets to filter organic material from the water (Flood andFiala-Meacutedioni 1982 Mackay and Wiggins 1979) Like otherextended phenotypes (see Glossary) orb spider threads and websexhibit physical and architectural plasticity (Blamires 2010Blamires et al 2014 2016 2017 Crews and Opell 2006Herberstein and Tso 2011 Scharf et al 2011 Townley et al2006 Tso et al 2007 Wu et al 2013) However viscous threadsare unusual in that after being spun they continue to exhibitplasticity as they respond to environmental conditions most notablyrelative humidity (RH) (see Glossary) (Agnarsson et al 2009Opell et al 2011a 2013 Sahni et al 2011 Stellwagen et al2015a 2014)

Temperature and ultraviolet light influence viscous threadproperties and performance (Stellwagen et al 2015b 20162014) although humidity has the greatest and most universaleffect As RH decreases during daylight hours temperatureincreases mediating the decrease in absolute humidity andreducing glycoprotein viscosity However species experience theimpact of humidity differently Orb weavers that live in exposedweedy vegetation experience greater daily oscillations in humiditythan those whose webs are anchored in vegetation that providesshade and helps maintain humidity (cf Fig 2AB) Although thefirst study of the effect of humidity on viscous thread adhesion waspublished over 30 years ago (Strohmenger and Nentwig 1987)renewed interest in this topic is revealing details about the impact ofhumidity on this complex and highly integrated natural adhesivesystem

Viscous capture thread productionUnderstanding the response of viscous threads to environmentalhumidity is key to understanding both the function and evolution ofthis unique adhesive system A viscous thread is a compound self-assembling adhesive produced by two aggregate gland spigotsflanking a conical flagelliform gland (see Glossary) spigot (Fig 1B)on each of a spiderrsquos paired posterior lateral spinnerets a total of sixspigots contributing to each thread (Coddington 1989 Park andMoon 2014 Peters 1955) As an axial line (see Glossary) emergesfrom the flagelliform spigotrsquos tip it is coated with aggregate glandsolution that contains glycoprotein and small hygroscopicmolecules (Townley and Tillinghast 2013) The coated axial

GlossaryAciniform glandsSpinning glands that produce large amounts of silk used to wrap andimmobilize preyAggregate glandOne of two spinning glands that open at the tips of adjacent spigots oneach posterior lateral spinneret (Fig 1B) and together coat a flagelliformfiber with a solution of inorganic salts and organic molecules which arereconfigured to form the outer lipid layer aqueous layer glycoproteincore and central granule of a viscous capture thread dropletAqueous layerThe material that covers a viscous threadrsquos axial lines and adhesiveglycoprotein cores This solution contains the low molecular masscompounds and inorganic salts that confer thread hygroscopicity andcondition and solvate the glycoproteins in the core of a viscous dropletThis layer is composed of aggregate glandmaterial that remains after theglycoprotein cores of droplets are formedAxial lines or axial fiberOne of two protein strands that is spun from a flagelliform gland spigot oneach posterior lateral spinneret and serves as one of the central supportlines of a viscous capture threadCribellate threadPlesiomorphic type of dry prey capture thread comprising an outer layerof thousands of nanofibrils that surround larger supporting fibersExtended phenotypeA physical product or construction of an animal that is geneticallydetermined affects its fitness and therefore can be shaped by naturalselectionFlagelliform glandA spinning gland that opens at the tip of a spigot found on each of theposterior lateral spinnerets (Fig 1B) and contributes one of the twosupporting axial lines of a viscous capture threadForaging humidityHumidity during the longest portion of an orb weaverrsquos feeding periodExcept for nocturnal species this corresponds to the times of lowerhumidity that occur from mid-morning to late afternoonGlycoproteinA polypeptide chain with attached carbohydrate groups These areconsidered the primary adhesives of viscous prey capture threadsLow molecular mass compounds (LMMCs)Small organic and inorganic molecules that are present in aggregategland secretions and remain in a viscous threadrsquos aqueous layer Theseare largely responsible for a viscous threadrsquos hygroscopicity and serve tosolvate and condition its glycoprotein adhesiveMajor ampullate glandSpinning gland that produces non-adhesive threads that form an orbwebrsquos attachment frame and radial lines (Fig 1A)PlateaundashRayleigh instabilityThe phenomenon by which the surface tension of the liquid in a thinstream or in the case of viscous threads a thin coating is lowered by theformation of small drops that minimize surface areaPyriform glandsSpinning glands that open in a cluster of spigots on a spiderrsquos anteriorlateral spinnerets and produce a dense zig-zag array of fibers that attachmajor ampullate threads to a substrateRelative humidity (RH)Water vapor pressure expressed as a percentage of maximum watervapor pressure at a given temperature and described by the formulaRH=(actual vapor pressuresaturated vapor pressure)times100Retention timeThe time an insect is retained by an orb webrsquos prey capture threadsbefore it can escapeSuspension bridge mechanismViscous threadrsquos ability to sum the adhesive forces generated bymultipledroplets as they extend transferring force to the threadrsquos flagelliformaxial lines which have assumed a parabolic configuration (Fig 7)Viscous capture threadThewet prey capture thread of araneoid spiders that features glycoproteinadhesive covered by a hygroscopic aqueous layer (Fig 1AD)

Youngrsquos modulusAlso referred to as elastic modulus describes the stiffness of a materialand is expressed as the energy per cross-sectional area required toextend a material Lower values denoting more easily stretched materialYoungrsquos modulus is determined as the slope of the linear region of amaterialrsquos stressndashstrain curve

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fibers (see Glossary) from each of the two spinnerets merge to forma single cylindrical thread after which PlateaundashRayleigh instability(see Glossary) causes the aggregate material to quickly form a seriesof evenly spaced droplets that exhibit a bead on a string (BOAS)morphology (Fig 1CD) (Edmonds and Vollrath 1992 Mead-Hunter et al 2012 Roe 1975) Environmental humidity affects thesize of the droplets that form through its impact on the viscosity ofthe aggregate material (Edmonds and Vollrath 1992 Sahni et al2012b) Studies of viscous thread analogs and droplet formation inthin films show that the velocity of thread production and the size andshape of nozzle apertures affect droplet spacing (Sadeghpour et al2017 Sahni et al 2012b) principles worth examining in viscousthread spinning At the center of each droplet a glycoprotein corecoalesces (Fig 1E) (Vollrath and Edmonds 1989) Although this isthe only droplet region where protein can be visualized under lightmicroscopy proteins are also found in the remaining aqueousmaterial which covers both the threadrsquos supporting axial fibers andits glycoprotein cores (Amarpuri et al 2015a)

Viscous capture thread structure and compositionFour droplet regions have been identified (1) a thin outer lipid coatfirst identified by Hans Peters (Peters 1995) and seen as a lsquoskinrsquo inscanning electron microscope images of desiccated droplets (Opelland Hendricks 2009) but poorly studied (2) the aqueous layer (seeGlossary) containing proteins and the small molecules that aredescribed in the following section (3) a distinct glycoprotein coreand (4) a granule in the corersquos center which is thought to anchor thecore to the threadrsquos flagelliform fibers (Opell and Hendricks 2010)Both the glycoprotein core and its granule are most clearly seenwhen a droplet has been flattened on amicroscope slide or coverslipEpi-illumination more clearly reveals the glycoprotein core

whereas the granule is more easily seen with transmitted lightwhere it appears as a cylinder or toroid within the core (Opell andHendricks 2010) Consequently in some older literature thegranule is assumed to be responsible for thread adhesion It is notknown if the granule is simply a region of the glycoprotein that hasbecome associated with flagelliform fibers or a distinct protein orproteins Although droplets resist being moved along the axialfibers they are not permanently bonded and can slide (Opell et al2011a 2013)

Despite the large percentage of water in a droplet the adhesion ofits glycoprotein is several orders of magnitude greater than thecapillary adhesion of its aqueous layer (Sahni et al 2010) Only onethread glycoprotein aggregate spider glue 2 or ASG2 has beencharacterized (Choresh et al 2009 Collin et al 2016Vasanthavada et al 2012) with ASG1 subsequently beingassociated with mucin proteins that bind chitin to cells (Collinet al 2016) Collin et al (2016) showed that ASG2 is a member ofthe spidroin gene family and suggested that consistent with spidroinnomenclature it be named aggregate spidroin 1 (AgSp1) Spidroinsare a class of scleroproteins that includes major ampullate andflagelliform fibers (Ayoub et al 2007 Garb et al 2010 2007Gatesy et al 2001) However the presence of AgSp1 proteins inglue droplets has not been confirmed and we do not know whetherAgSp1 is the only glycoprotein gene or if this type of protein is theonly adhesive in a droplet The challenge of adhering to an insectrsquoswaxy epicuticle is great and our understanding of AgSp1rsquos mode ofadhesion is poor relative to that of other bioadhesives such asmussel glue (Forooshani and Lee 2017) Although glycoproteinsare known to be adhesives (eg von der Mark and Sorokin 2002Xu and Mosher 2011) until information about possiblepost-translational modifications of AgSp1 proteins and their

A

C

D

E

200 microm 30 microm

VCT

RT10 mm

B

40 microm

Fig 1 Viscous capture thread production andcomposition (A) A female Argiope aurantia spinsa viscous capture thread (VCT) prior to attachingit to a major ampullate radial thread (RT)(B) Scanning electron microscope image of thespinning spigots on one posterior lateral spinneretthat are responsible for producing a viscous capturethread AG aggregate gland spigots FLflagelliform gland spigot (C) An Argiope trifasciatathread showing droplets forming from the aggregatematerial cylinder (D) The same thread less than30 s later after droplets have formed (E) ANeoscona crucifera droplet that has been flattenedagainst a glass coverslip at 90 relative humidity toshow its glycoprotein core attached to flagelliformaxial fibers and surrounded by aqueous materialPanel B adapted from Blackledge et al (2009a)

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three-dimensional structure is available it will be difficult todetermine their mode of adhesion

Viscous thread response to humidity in a spiderrsquosenvironmentViscous glue droplets contain abundant amounts of water-solubleorganic and inorganic compounds that are hygroscopic in natureThese are often termed low molecular mass compounds(LMMCs) (see Glossary) Most are organic and only 10ndash20are inorganic compounds (Townley and Tillinghast 2013)Organic LMMCs are small polar aliphatic compounds (mostlyamine and sulphate based) such as alanine choline betaineproline glycine taurine GABamide putrescine N-acetyltaurineN-acetylputrescine and isethionic acid (Fig 3) (Anderson andTillinghast 1980 Tillinghast et al 1987 Townley et al 1991

2012 2006 Townley and Tillinghast 2013 Vollrath et al 1990)Inorganic LMMCs include H2PO4

minus K+ NO3minus Na+ Clminus and Ca2+

moieties (Anderson and Tillinghast 1980 Townley andTillinghast 2013 Townley et al 2006 Vollrath et al 1990)

The LMMCs are hypothesized to have evolved in part fromneurotransmitters (Edmonds and Vollrath 1992) but are nowdistributed throughout the aqueous material where they function totake up water from the environment and interact with glycoproteinsto render the glue functional in different humidity conditions(Amarpuri et al 2015b Opell et al 2013 Sahni et al 2011 2014Townley and Tillinghast 2013) Individual LMMCs differ widelyin hygroscopic response Compounds such as choline and N-acetyltaurine are hygroscopic over a range of humidity conditionsGABamide N-acetylputrescine and isethionic acid start adsorbingat approximately 55 RH whereas glycine potassium nitrate and

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Date18 Aug 5 Sep

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) C155

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copr

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eav

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m2 microm

3 )

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F25

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Fig 2 Daily changes in environmental humidity and its effect on viscous thread properties and insect retention time (A) Daily changes in relativehumidity (RH) in the exposed weedy vegetation habitat ofArgiope aurantia during 2011 (B) RH and temperature in the forest edge habitat ofAraneus marmoreusfrom 15 August to 15 October 2016 (C) Mean absolute humidity in this A marmoreus habitat (D) Volume-specific glycoprotein flattened area (solid circles) andextension (open circles) at five humidities (E) Change in the relative work required to extend the droplets of a 4 mm thread span to the initiation of pull-off at fivehumidities (F) Active struggle time required by a housefly to escape from three capture thread strands showing the association of viscous droplet andthread features with insect retention time Images to the right of panels DndashF illustrate the properties that are plotted Error bars are plusmn1 se Panels DndashF areobservations made at 23degC Panel A is adapted from Opell et al (2013) and panels BndashF from Opell et al (2017)

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potassium dihydrogen phosphate show less than 3 water uptakeby mass even at high humidity conditions (Townley et al 1991Vollrath et al 1990) LMMCs differ in their types andcompositions across orb-weaving species living in habitats withdifferent humidity levels (Fig 3) However it is important to notethat even among individuals of the same species LMMCscomposition differs and is presumed to be affected by a spiderrsquosgenetics and diet (Higgins et al 2001)The primary function of the LMMCs is to solvate and soften

glycoproteins to enhance adhesion The LMMCs interact with theglycoproteins to make viscid glue functionally responsive tohumidity in the environment Pristine thread droplets swell as RHincreases whereas removal of the hygroscopic compounds bywashing threads with water leads to the collapse of the glue structureand renders it incapable of subsequently taking up more than10ndash20water even at high humidity After this collapse it becomesimpossible to reintroduce LMMCs back into the washed glue torecover adhesion and at 100 RH washed threads lose two ordersof magnitude of adhesion compared with pristine threads(Fig 4AB) In all conditions (0 40 100 RH or wet)washed glue droplets fail to make intimate contact and do not adhereto the surface (Sahni et al 2014) Various solid-state nuclearmagnetic resonance (NMR) spectroscopy techniques have shownthat the glycoproteins soften and become humidity responsive in thepresence of LMMCs Cross-polarization magic-angle spinning(CPMAS) NMR is sensitive to rigid molecules and demonstratesthat the rigidity of glycoproteins in pristine glue decreases ashumidity is increased from 0 RH to 100 RH (indicated by thedecrease in intensity of the spectrum in Fig 4C) This directlycorrelates with macro-level observations of glue getting softer ashumidity rises resulting in intimate contact with surfaces and

enhanced adhesion When LMMCs are washed off the viscid glueis irresponsive to humidity (Fig 4D) and the glycoproteins becomerigid corresponding to the collapse of the glue at a macro level(Sahni et al 2014) Altering LMMCs composition provides amechanism by which natural selection can optimize viscous threadperformance to the humidity in a speciesrsquo environment

Viscous droplet volume responds dramatically to changes inhumidity (Fig 5A) (Opell et al 2011a 2013) However as we willexplain the degree of droplet hygroscopicity differs among speciesand is related to the humidity of a speciesrsquo habitat Glycoproteinvolume also responds to humidity (Fig 5C) documenting that afteratmospheric water enters a dropletrsquos aqueous layer some of it isabsorbed by the glycoprotein core This results in an increase indroplet extensibility as humidity increases (Fig 5B) Even afterextension is adjusted for glycoprotein volume this response differsamong species (Fig 5DE) Compared with the lower hygroscopicdroplets of species such as Neoscona crucifera and Verrucosaarenata that occupy humid environments the more hygroscopicdroplets of Argiope aurantia and Larinioides cornutus do not extendas far at higher humidities before releasing because their glycoproteinmore easily becomes over lubricated dropping in viscosity and moreeasily releases from a surface (Fig 5D) (Opell et al 2013 Sahniet al 2011) Thus the viscosity of A aurantia glycoprotein at 55RH is similar to that ofN crucifera at 90RH (Fig 5DE) Althoughthe greater hygroscopicity of A aurantia threads might appear to be adeficiency it is in fact an adaptation to remaining hydrated duringthe late morning and afternoon hours when humidity is low (Fig 2A)

The level of humidity at which adhesion of viscid glues reaches amaximum in different spider species corresponds to their foraginghabitats (Fig 6A) Maximum adhesion occurs when the viscosity ofthe glue is such that the contribution of two factors is optimized

A B

C D

Nocturnal

Neoscona crucifera

Forest edge

GABamide

Alanine

Glycine

Choline

N-Acetyltaurine

Putrescine

lsethionic acid

N-Acetylputrescine

Betaine

Taurine

Proline

Humidity

Araneus marmoreus

Forest interior

Verrucosa arenata

Open fieldsLow

Argiope aurantia

High

145

2

49

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12

4

11

108

29

7

1025

15

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9

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86

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Fig 3 Diversity of organic low molecularmass compounds (LMMCs) in viscid glues oforb web spiders (AndashD) Relative compositions ofdiverse organic LMMCs (color coded as depictedin key) present in the glues of orb webs belongingto Neoscona crucifera Araneus marmoreusVerrucosa arenata and Argiope aurantia eachinhabiting a habitat with a different foraginghumidity (see Glossary) Not only do thepercentage compositions of LMMCs such asGABamide and choline differ among species butsome LMMCs are restricted to certain speciesFor example taurine is found only in A aurantiaisethionic acid is found only in A marmoreus andA aurantia and betaine is present in all speciesbut A marmoreus These differences areexplained by many factors that probably includethe hygroscopic strength of the LMMCs theirmetabolic costs competition for thesecompounds across metabolic processes andphylogenetic relationship among the speciesrepresented The effect on each speciesrsquo uniquemix of LMMCs on droplet hygroscopicity is shownin Fig 5C and on thread adhesion at differenthumidities in Fig 6A

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surface interactions (substratendashglue interaction energy andspreading area) and bulk dissipation (rate of peeling andviscosity) (Amarpuri et al 2015b) As RH increases spreadingof the droplets improves as bulk dissipation decreases (Fig 6B) Atlow humidity droplets are stiff and do not spread efficiently Ashumidity increases droplets spread and resist peeling as theglycoprotein extends leading to generation of high adhesiveforces At high humidity droplets coalesce to form a sheet of gluethat spreads completely but breaks easily These changes inbehavior represent a remarkable 1000-fold variation in glueviscosity but adhesion is maximized in a relatively narrow rangeof viscosity that optimizes spreading and bulk contributions(Fig 6C) Remarkably this optimal viscosity is achieved at verydifferent humidities in different species that closely matches whereeach forages (Fig 6A) Thus the diverse mixture of LMMCs(Fig 3) adapts species to a range of habitat humidities (Amarpuriet al 2015b Opell et al 2015 2013) In the next section weexplain why maintaining glycoprotein extensibility plays animportant role in thread adhesion

Summing the adhesive forces of individual dropletsIn the milliseconds after an insect strikes a web a viscous capturethreadrsquos glycoprotein cores must spread immediately to establishadhesion and then as the insect struggles to escape instantly resistshifting forces that threaten to pull threads from the insectrsquos bodyand wings If the axial lines and droplets were rigid force applied toa thread would cause the terminal droplets to release and initiateserial droplet pull-off that would quickly lead to thread releaseCompared with cribellate thread the plesiomorphic dry preycapture threads spun by araneoid ancestors (Garrison et al 2016)viscous thread is more effective in this regard Cribellate threads areformed of several thousand dry protein nanofibers arrayed aroundsupport lines and can adhere by van der Waals forces capillaryattachment snagging on insect setae (Joel et al 2015 Opell 2013)and can even embed their nanofibrils in the waxy outer epicuticle of

an insectrsquos exoskeleton (Bott et al 2017) Although versatile theadhesion of this thread is limited by the stiffness of its internalsupporting fibers Its adhesion does not increase as increasinglengths of thread contact a surface indicating that after the adhesionof terminal thread regions fails crack propagation ensuespreventing additional adhesion being recruited from more centralthread regions (Opell and Schwend 2008)

In contrast viscous thread adhesion increases as the threadcontact length increases (Opell and Hendricks 2007 2009) Thepliable adhesive droplets of viscous threads combine with thethreadrsquos extensible flagelliform support lines (Blackledge andHayashi 2006) to create a dynamic adhesive system that assumesthe configuration of a lsquosuspension bridgersquo as it sums the adhesiveforces of multiple droplets (Fig 7) Moreover as force is applied toa thread the extension of its droplets and flagelliform linescombines to dissipate the energy of a struggling prey (Piorkowskiand Blackledge 2017 Sahni et al 2011) Thus there are two waysto characterize viscous thread adhesion the force required to pull athread from a surface (eg Opell and Hendricks 2007 2009) andthe work of adhesion required to bring a thread to the point of pull-off (eg Sahni et al 2011)

The threadrsquos hygroscopic aqueous layer also makes an essentialcontribution to the suspension bridge mechanism (see Glossary) byensuring that flagelliform fibers remain hydrated and extensibleWhen threads were stretched experimentally to reduce axial fiberextensibility but the number of contributing droplets wasmaintained by contacting longer thread lengths the force requiredto pull a thread from a surface decreased (Opell et al 2008)Flagelliform fiber extension is also crucial for a threadrsquos ability todissipate the energy of a struggling insect (Sahni et al 2011)contributing more than twice the work of adhesion as combineddroplet extensions (Piorkowski and Blackledge 2017)

Because viscous threads rely on the extensibility of bothflagelliform fibers and the glycoprotein cores of droplets theperformance of these two components must have evolved in a

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Glycoprotein Glycoprotein

Aromatic Aromatic

Aliphatic Aliphatic

120

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50 0 200 150 100 50 0

110 100 90 80 120 110 100 90 80

ndashC=O ndashC=O

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kine

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kine

ss (micro

N)

Wwet W100 W100 P100

04

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Fig 4 Interaction of lowmolecular masscompounds (LMMCs) and glycoproteinsin adhesion of viscid threads(AB) Adhesion forces for pristine (P) andwashed (W obtained after removal ofLMMCs) capture silk threads of Larinioidescornutus tested on glass substrates underdifferent conditions [P0 W0 desiccatedP100 W100 100 relative humidity (RH)W40 40 RH Wwet externally wetted](CD) Cross-polarization magic-anglespinning solid-state nuclear magneticresonance measurements for pristine(C) and washed (D) capture silk threads ofL cornutus recorded at 0RH (blue) 35RH (green) and 100 RH (red) Adaptedand reprinted with permission from SahniV Miyoshi T Chen K Jain D BlamiresS J Blackledge T A and Dhinojwala A(2014) Direct solvation of glycoproteins bysalts in spider silk glues enhancesadhesion and helps to explain theevolution of modern spider orb websBiomacromolecules 15 1225-1232Copyright 2014 American ChemicalSociety

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complementary fashion If glycoprotein is too stiff relative to athreadrsquos flagelliform fibers the outer droplets of a contacting strandwill release before inner droplets have extended and contributedtheir adhesive forces If by contrast glycoprotein extensibility is too

great there will be little resistance and the axial line will bowacutely with little work being done and little adhesive force beingsummed This is borne out by a comparison of the Youngrsquosmodulus (see Glossary) of three speciesrsquo flagelliform fibers and

Fig 5 The effect of humidity on viscous thread droplet volume glycoprotein volume and droplet extensibility at 23degC (A) The same Argiope aurantiadroplet imaged at three relative humidities (B) The impact of relative humidity on the extensibility of A aurantia droplets (C) Increases in droplet and glycoproteinvolumes of five orb weavers that occupy different habitats (D) The extension of A aurantia droplets at different humidities relative to a dropletrsquos glycoproteinvolume (E) The extension ofN crucifera droplets at different humidities relative to a dropletrsquos glycoprotein volume Above 55 relative humidity (RH) A aurantiaglycoprotein becomes over lubricated causing it to pull from a surface before its full extension is expressed In contrastN crucifera droplets attract less moisturecausing glycoprotein viscosity to decrease and extension to increase but never absorb enough moisture to become over lubricated Diagrams below panels Dand E depict this decrease in a glycoprotein viscosity with increasing humidity as seen in a dropletrsquos contact footprint that is circled on the left of each series Errorbars are plusmn1 se Adapted from or constructed from data in Opell et al (2013) and BDO unpublished

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glycoproteins Youngrsquos modulus (E) is a measure of a materialrsquosstiffness with smaller values indicating a material that is moreeasily extended When compared at 50 RH flagelliform E rangedfrom 0009 to 00300 GPa and glycoprotein E from 000003 to00014 GPa with flagelliform E being 21 52 and 290 times greaterthan glycoprotein E for the three species (BDO M E Clouse andS F Andrews unpublished Sensenig et al 2010)

Physiological and ecological impact of humidityAs the studies of Tillinghast Townley Vollrath and their colleagueshave shown (Edmonds and Vollrath 1992 Townley et al 19912012 2006 Townley and Tillinghast 2013 Vollrath et al 1990Vollrath and Tillinghast 1991) environmental humidity plays acrucial role in the function of an orb web from the time that it isconstructed until it is taken down and its silk ingested Highhumidity during the later evening and early morning hours whenmost orb webs are constructed affects the self-assembly of the gluedroplets of viscous capture threads Changes in humidity over thecourse of a day (Fig 2AndashC) affect thewebrsquos ability to bothwithstandprey impact (Boutry and Blackledge 2013) and retain interceptedprey (Opell et al 2017) Finally when ingested the fully hydratedglue droplets supply a spider with both water and recyclablenutrients (Edmonds and Vollrath 1992 Townley and Tillinghast1988) In fact some important LMMCs like choline are also

necessary for spider physiology and are in short supply beingobtained only from insect prey and ingested threads (Higgins andRankin 1999 Townley and Tillinghast 2013 Townley et al 2006)

As we gain a greater understanding of viscous threadhygroscopicity and fine-scale humidity-mediated changes inviscous droplets it is important to determine how these featuresimpact prey retention time because this is ultimately how naturalselection must tune thread performance to the humidity of a speciesrsquoenvironment However assessing prey retention particularly invertically oriented orb webs like most of those that have beenstudied is challenging Retention is affected by many factorsincluding the mass of an insect and its impact velocity the numberof capture threads that it strikes the texture of the insectrsquos bodyregion that contacts a thread the region of the web a prey strikes andwhether after struggling free from these threads the insect tumblesinto other capture threads (Blackledge and Zevenbergen 2006Opell and Schwend 2007 Sensenig et al 2013 Zschokke andNakata 2015)

To make humidity the focal variable an anesthetized houseflywas placed wings downward across three equally spacedhorizontal capture thread strands from the large orb weaverAraneus marmoreus (Fig 2F) and its escape captured in a videorecording (Opell et al 2017) The humidity maximizing retentiontime of the flies was predicted to be the humidity at which both the

A B

C

Tetragnatha

30

0 s 01 s 1 s

50

70

90

7

4

3

2

2


Wor

k do

ne d

urin

g pe

elin

g (n

orm

aliz

ed J

)

70 80 90

Neoscona

Larinioides

Verrucosa

Argiope

Humidity

Viscosity

Hum

idD

ryFo

ragi

ng h

abita

t hum

idity

Bul

k di

ssip

atio

nasymp

resi

stan

ce to

def

orm

atio

n

Spr

edin

g asymp

surfa

ce c

onta

ct a

rea

Adh

esio

n

Fig 6 Tuning viscous thread to habitat humidity (A) Maximum adhesion response as a function of humidity for capture silk threads belonging to speciesoccupying different habitat humidities (B) Progressive spreading of Larinioides cornutus glycoprotein glue (left to right) under conditions of low (top) to high(bottom) humidity Scale bar 50 microm (C) Diagram showing how glycoprotein spreading (red) and bulk dissipation or viscosity (green) trends must be balanced toproduce an optimized adhesion response Adapted and reprinted with permission from Amarpuri G Zhang C Diaz C Opell B D Blackledge T A andDhinojwala A (2015) Spiders tune glue viscosity to maximize adhesion ASC Nano 9 11472-11478 Copyright 2015 American Chemical Society

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surface area and extensibility of the glycoprotein were greatest(Fig 2D) This occurred at 72 RH the same level at which theenergy estimated to bring a 4 mm span of capture thread to theinitiation of pull-off was greatest and thus most difficult for a preyto achieve (Fig 2E) This humidity is also similar to the afternoonhumidity at the forest edge where A marmoreus lives (Fig 2B) At72 RH actively struggling flies were retained 11 s longer than ateither 37 or 55 RH (Fig 2F) This additional time isecologically significant because it provides a spider more time tolocate and reach an insect and to begin wrapping it with silk fromnumerous aciniform gland (see Glossary) spigots on the posteriormedian and posterior lateral spinnerets (Coddington 1989Tremblay et al 2015) before the prey can escape the webGreater retention times also relate directly to the size of insects

that a web can retain For large orb weavers such as A marmoreus itis postulated that these large rare prey are more profitable andcomprise the greatest proportion of a spiderrsquos total food intake(Blackledge 2011 Venner and Casas 2005) but see Eberhard(Eberhard 2013) for challenges to this hypothesis Thus there issolid evidence that longer prey retention time selects for changes inthe composition of a viscous threadrsquos hygroscopic compounds thattune thread performance to the humidity of a speciesrsquo habitat Thesefindings are the first step in ascribing fitness values to theperformance characteristics of viscous threads As data for otherspecies are added it should be possible to rank the relativecontributions of glycoprotein surface area viscosity and extensionto prey retention time

Synthetic viscous threads as models for adhesivesHumidity poses serious problems to the stability of adhesive joints(Abdel Wahab 2012 Brewis et al 1990 Petrie 2007 Tan et al2008 White et al 2005) Most of the synthetic adhesives fail when acrucial RH is exceeded (Petrie 2007 Tan et al 2008) Therefore itwould be desirable to have synthetic adhesives that can either resistchanges in RH and continue to strongly bind surfaces or respondwith

humidity similar to viscid silk The unique natural designs of bothcribellate and viscous prey capture threads have inspired researchersto develop similarly structured materials for a variety of applicationsincluding adhesives water collectors and solidndashliquid hybridmaterials (Bai et al 2012 Chen and Zheng 2014 Elettro et al2016 Sahni et al 2012b Song et al 2014 Tian et al 2011) In oneof the first attempts synthetic adhesive BOAS microthreads werefabricated by drawing a synthetic nylon thread through a pool ofpolydimethylsiloxane (PDMS) polymer (Sahni et al 2012b) Theprocess created a cylindrical coating that formed smaller droplets dueto PlateaundashRayleigh instability and these threads were sticky whentested on a glass substrate (Fig 8) The spacing and diameter of thesesynthetic thread droplets were varied by changing the capillarynumber (Ca=velocitytimesviscositysurface tension) which depends ondrawing velocity PDMS viscosity and surface tension (Fig 8AndashC)A higher capillary number (higher velocity higher viscosity andlower surface tension) produced larger and more widely spaceddroplets (Fig 8C) which exhibited greater adhesion (Fig 8E) Thestudy presented a simple and effective manner of creating BOASadhesive mimics of viscous threads (Fig 8D) and also helped intesting the fundamental principles behind the adhesion of viscid silkby using synthetic mimics (Sahni et al 2012b) This successfulstrategy can also be used to generate humidity-responsive adhesivesFor example droplets can be laden with mixtures of LMMCsmimicking natural compositions (Fig 3) incorporated withinpolymer matrices to generate viscous thread to synthesizehumidity-sensitive adhesives These synthetic adhesive structurescan then be used in applications such as a bandages or adhesive tapeswhere adhesion is crucial in the presence of water

Fig 7 A single Verrucosa arenata capture thread being pulled from a2 mm wide contact plate Adhesive forces from the threadrsquos progressivelyextending droplets are summed by being collectively transferred to thedeflected axial line In the top frame a droplet near the strandrsquos center hasreleased from the plate introducing an instability that will initiate adhesivefailure

A B C E

D150 microm

01

0

10

20

30

02Capillary no

Adh

esio

nen

ergy

(10

ndash3 micro

J)

03

Fig 8 Synthetic adhesive threads and their performance (AndashC) Adhesivepolydimethylsiloxane (PDMS) microthreads with differences in droplet spacingand diameter resulting from differences in the velocity with which nylon threadswere drawn through a PDMS solution (D) Image showing the formation of asuspension bridge when a synthetic microthread is pulled from a glasssubstrate (E) Variation in adhesive energy generated during pull-off ofsynthetic microthread with different capillary numbers Adapted and reprintedwith permission from Sahni V Labhasetwar D V and Dhinojwala A (2012)Spider silk inspired functional microthreads Langmuir 28 2206-2210Copyright 2012 American Chemical Society This shows that it is possible tofabricate microthreads that in many ways mimic the appearance andperformance of spider viscous threads

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Conclusions and outlookViscous thread adhesion relies heavily on water for both effectivespreading of the adhesive glycoproteins and elasticity of theunderlying axial thread Water content also influences the PlateaundashRayleigh instability that determines the final size and spacing of gluedroplets These features act synergistically to generate substantialadhesion as viscous threads deform in a suspension bridge-likepattern while detaching from a variety of surfaces Some of this watercan be obtained directly from the atmosphere when threads are firstspun potentially resulting in a net gain of water by a spider when anorb web is taken down and its silk ingested Most orb webs are spununder humid conditions in the late evening or early morning so thatminimal hygroscopicity is likely to be necessary for dropletformation and adhesion (Blackledge et al 2009a) However wehypothesize that increased thread hygroscopicity was necessary tooptimize thread adhesion as orb weavers diversified to occupyhabitats where humidity drops during the course of a day Thusnatural selection tuned the composition of LMMCs in a dropletrsquosouter aqueous layer to meet this challenge (Townley and Tillinghast2013) and to maintain glycoprotein structure and enhance its surfaceinteractions (Liao et al 2015) However this is largely based oninvestigation of a few temperate species of spiders and three keyquestions remain about viscid thread hygroscopicity First whatabout species in consistently arid or humid habitats such as desertsand rainforests Do their glues perform similarly or show distinctLMMCs compositions Second can individual spiders controlLMMCs composition physiologically to tailor thread structure andadhesion under different physiological conditions Finally did thehygroscopicity system arise to help spiders conserve waterresources after viscid glue was already being produced (eg theancestral condition was for orb spiders to exude wet sticky secretionsfrom their aggregate glands) or as a mechanism to improve adhesion(Opell et al 2011b Piorkowski and Blackledge 2017) with spidersadding LMMCs to dry adhesive secretions for some other functionalbenefitOur current model of the evolution of viscous thread

environmental responsiveness relies entirely on describingvariation in LMMCs composition The amino acid sequence ofonly one glycoprotein has been characterized and details of thismoleculersquos three-dimensional structure and adhesion are not wellunderstood Thus the model we present here is clearly anoversimplified view For instance how much of the variation inthe environmental responsiveness of different speciesrsquo glue isexplained by interactions between LMMCs and variation inglycoprotein sequence Future investigation should also focus onunderstanding how LMMCs directly interact the glycoproteins toplasticize them and how this influences adhesion Indeed selectionfor optimal glycoprotein secondary structure may be as important asselection for optimal aqueous layer hygroscopicityThe use of LMMCs to recruit water and control the self-

organization of a hierarchically structured adhesive thread is simplein concept and therefore translatable to synthetic models Howeverwe still do not understand the specific functions of individualLMMCs and the mechanisms by which they plasticize the adhesiveglycoproteins In addition to optimizing the performance ofsynthetic adhesives such research will also provide a powerfultool to test hypotheses about specific aspects of viscous threadfunction and spider web evolution

AcknowledgementsWe are grateful to two reviewers whose comments and suggestions allowed us toimprove the clarity and completeness of this Review

Competing interestsThe authors declare no competing or financial interests

FundingNational Science Foundation grant IOS-1257719 supported our research on viscousthread hygroscopicity and the preparation of this Review

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Venner S and Casas J (2005) Spider webs designed for rare but life-savingcatches Proc R Soc B 272 1587-1592

Vollrath F (2005) Spidersrsquo webs Curr Biol 15 R364-R365Vollrath F and Edmonds D (1989) Modulation of the mechanical properties of

spider silk coating with water Nature 340 305-307Vollrath F and Selden P (2007) The role of behavior in the evolution of spiders

silks and webs Annu Rev Ecol Evol Syst 38 819-846Vollrath F and Tillinghast E K (1991) Glycoprotein glue beneath a spider webrsquos

aqueous coat Naturwissenschaften 78 557-559Vollrath F Fairbrother W J Williams R J P Tillinghast E K Bernstein

D T Gallagher K S and Townley M A (1990) Compounds in the droplets ofthe orb spiderrsquos viscid spiral Nature 345 526-528

von der Mark K and Sorokin L (2002) Adhesive Glycoproteins In ConnectiveTissue and Its Heritable Disorders Molecular Genetic and Medical Aspects 2ndedn (ed P M Royce and B Steinmann) pp 293-328 New York Wiley-Liss Inc

Waite J H (2017) Mussel adhesionndashessential footwork J Exp Biol 220517-530

White C Tan K T Hunston D Steffens K Stanley D L Satija S KAkgun B and Vogt B D (2005) Mechanisms of criticality in environmentaladhesion loss Soft Mat 11 3994-4001

Wolff J O Grawe I Wirth M Karstedt A and Gorb S N (2015) Spiderrsquossuper-glue thread anchors are composite adhesives with synergistic hierarchicalorganization Soft Mat 11 2394-2403

World Spider Catalog (2017) World Spider Catalog v185 httpwscnmbechWu C C Blamires S J Wu C L and Tso I M (2013) Wind induces variations

in spider web geometry and sticky spiral droplet volume J Exp Biol 2163342-3349

Xu J and Mosher D (2011) Fibronectin and Other Adhesive Glycoproteins InThe Extracellular Matrix an Overview (ed R P Mecham) pp 41-75 New YorkSpringer

Zschokke S and Nakata K (2015) Vertical asymmetries in orb websBiol J Linn Soc 114 659-672

12


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ofEx

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iology

2012) while spirally arrayed adhesive prey capture threadsproduced from flagelliform and aggregate glands (see Glossary)retain the insect (Sahni et al 2013) until the spider can locate run toand begin to subdue it Material invested in non-adhesive threadsinfluences the size and velocity of insects that a web can stop(Sensenig et al 2012) and material invested in capture threadaffects the time an insect is trapped (Opell et al 2017) A retentiontime (see Glossary) difference of even a few seconds can be thedifference between a prey being captured or lost (Eberhard 1989)

Orb-weaving spiders are not unique in relying on extendedphenotypes (see Glossary) for important functions (Dawkins1982) nor are they the only animals that use these products forprey capture For example parchment worms and caddisfly larvaeemploy nets to filter organic material from the water (Flood andFiala-Meacutedioni 1982 Mackay and Wiggins 1979) Like otherextended phenotypes (see Glossary) orb spider threads and websexhibit physical and architectural plasticity (Blamires 2010Blamires et al 2014 2016 2017 Crews and Opell 2006Herberstein and Tso 2011 Scharf et al 2011 Townley et al2006 Tso et al 2007 Wu et al 2013) However viscous threadsare unusual in that after being spun they continue to exhibitplasticity as they respond to environmental conditions most notablyrelative humidity (RH) (see Glossary) (Agnarsson et al 2009Opell et al 2011a 2013 Sahni et al 2011 Stellwagen et al2015a 2014)

Temperature and ultraviolet light influence viscous threadproperties and performance (Stellwagen et al 2015b 20162014) although humidity has the greatest and most universaleffect As RH decreases during daylight hours temperatureincreases mediating the decrease in absolute humidity andreducing glycoprotein viscosity However species experience theimpact of humidity differently Orb weavers that live in exposedweedy vegetation experience greater daily oscillations in humiditythan those whose webs are anchored in vegetation that providesshade and helps maintain humidity (cf Fig 2AB) Although thefirst study of the effect of humidity on viscous thread adhesion waspublished over 30 years ago (Strohmenger and Nentwig 1987)renewed interest in this topic is revealing details about the impact ofhumidity on this complex and highly integrated natural adhesivesystem

Viscous capture thread productionUnderstanding the response of viscous threads to environmentalhumidity is key to understanding both the function and evolution ofthis unique adhesive system A viscous thread is a compound self-assembling adhesive produced by two aggregate gland spigotsflanking a conical flagelliform gland (see Glossary) spigot (Fig 1B)on each of a spiderrsquos paired posterior lateral spinnerets a total of sixspigots contributing to each thread (Coddington 1989 Park andMoon 2014 Peters 1955) As an axial line (see Glossary) emergesfrom the flagelliform spigotrsquos tip it is coated with aggregate glandsolution that contains glycoprotein and small hygroscopicmolecules (Townley and Tillinghast 2013) The coated axial

GlossaryAciniform glandsSpinning glands that produce large amounts of silk used to wrap andimmobilize preyAggregate glandOne of two spinning glands that open at the tips of adjacent spigots oneach posterior lateral spinneret (Fig 1B) and together coat a flagelliformfiber with a solution of inorganic salts and organic molecules which arereconfigured to form the outer lipid layer aqueous layer glycoproteincore and central granule of a viscous capture thread dropletAqueous layerThe material that covers a viscous threadrsquos axial lines and adhesiveglycoprotein cores This solution contains the low molecular masscompounds and inorganic salts that confer thread hygroscopicity andcondition and solvate the glycoproteins in the core of a viscous dropletThis layer is composed of aggregate glandmaterial that remains after theglycoprotein cores of droplets are formedAxial lines or axial fiberOne of two protein strands that is spun from a flagelliform gland spigot oneach posterior lateral spinneret and serves as one of the central supportlines of a viscous capture threadCribellate threadPlesiomorphic type of dry prey capture thread comprising an outer layerof thousands of nanofibrils that surround larger supporting fibersExtended phenotypeA physical product or construction of an animal that is geneticallydetermined affects its fitness and therefore can be shaped by naturalselectionFlagelliform glandA spinning gland that opens at the tip of a spigot found on each of theposterior lateral spinnerets (Fig 1B) and contributes one of the twosupporting axial lines of a viscous capture threadForaging humidityHumidity during the longest portion of an orb weaverrsquos feeding periodExcept for nocturnal species this corresponds to the times of lowerhumidity that occur from mid-morning to late afternoonGlycoproteinA polypeptide chain with attached carbohydrate groups These areconsidered the primary adhesives of viscous prey capture threadsLow molecular mass compounds (LMMCs)Small organic and inorganic molecules that are present in aggregategland secretions and remain in a viscous threadrsquos aqueous layer Theseare largely responsible for a viscous threadrsquos hygroscopicity and serve tosolvate and condition its glycoprotein adhesiveMajor ampullate glandSpinning gland that produces non-adhesive threads that form an orbwebrsquos attachment frame and radial lines (Fig 1A)PlateaundashRayleigh instabilityThe phenomenon by which the surface tension of the liquid in a thinstream or in the case of viscous threads a thin coating is lowered by theformation of small drops that minimize surface areaPyriform glandsSpinning glands that open in a cluster of spigots on a spiderrsquos anteriorlateral spinnerets and produce a dense zig-zag array of fibers that attachmajor ampullate threads to a substrateRelative humidity (RH)Water vapor pressure expressed as a percentage of maximum watervapor pressure at a given temperature and described by the formulaRH=(actual vapor pressuresaturated vapor pressure)times100Retention timeThe time an insect is retained by an orb webrsquos prey capture threadsbefore it can escapeSuspension bridge mechanismViscous threadrsquos ability to sum the adhesive forces generated bymultipledroplets as they extend transferring force to the threadrsquos flagelliformaxial lines which have assumed a parabolic configuration (Fig 7)Viscous capture threadThewet prey capture thread of araneoid spiders that features glycoproteinadhesive covered by a hygroscopic aqueous layer (Fig 1AD)

Youngrsquos modulusAlso referred to as elastic modulus describes the stiffness of a materialand is expressed as the energy per cross-sectional area required toextend a material Lower values denoting more easily stretched materialYoungrsquos modulus is determined as the slope of the linear region of amaterialrsquos stressndashstrain curve

2


Journal

ofEx

perim

entalB

iology





A

C

D

E


VCT

RT10 mm

B

40 microm


3


Journal

ofEx

perim

entalB

iology







Rel

ativ

e hu

mid

ity (

) 90

70

50

30

A

Date18 Aug 5 Sep

Rel

ativ

e hu

mid

ity (

)

Tem

pera

ture

(degC

)

Time (h)

B100

95

90

8 10 12 14 16 18 20

85

80

75

70

65

23

24

22

21

20

19

18

17

16

Abs

olut

e hu

mid

ity (g

mndash3

) C155

15

145

14

1358 10 12 14 16 18 20

Gly

copr

otei

n ar

eav

olum

e (micro

m2 microm

3 )

Leng

thg

lyco

prot

ein

volu

me

(microm

microm

3 )

D03

025

02

015

01

08

07

06

05

04

03

02

01

0


80 100

Rel

ativ

e w

ork

ofdr

ople

t ext

ensi

on

E250

200

150

100

50

Inse

ct re

tent

ion

time

(s)

F25

20

15

10

5


4


Journal

ofEx

perim

entalB

iology






A B

C D

Nocturnal

Neoscona crucifera

Forest edge

GABamide

Alanine

Glycine

Choline

N-Acetyltaurine

Putrescine

lsethionic acid

N-Acetylputrescine

Betaine

Taurine

Proline

Humidity

Araneus marmoreus

Forest interior

Verrucosa arenata

Open fieldsLow

Argiope aurantia

High

145

2

49

6

38

19

12

4

11

108

29

7

1025

15

6

6

11

21

6

9

4

18

86

15

14

12


5


Journal

ofEx

perim

entalB

iology







10 A B

C D

8

6

4

2

0P0 W0 W40

Conditions


Aromatic Aromatic

Aliphatic Aliphatic

120


50 0 200 150 100 50 0

110 100 90 80 120 110 100 90 80

ndashC=O ndashC=O

Stic

kine

ss (m

N)

Stic

kine

ss (micro

N)

Wwet W100 W100 P100

04

03

02

01

0


6


Journal

ofEx

perim

entalB

iology




7


Journal

ofEx

perim

entalB

iology






A B

C

Tetragnatha

30

0 s 01 s 1 s

50

70

90

7

4

3

2

2


Wor

k do

ne d

urin

g pe

elin

g (n

orm

aliz

ed J

)

70 80 90

Neoscona

Larinioides

Verrucosa

Argiope

Humidity

Viscosity

Hum

idD

ryFo

ragi

ng h

abita

t hum

idity

Bul

k di

ssip

atio

nasymp

resi

stan

ce to

def

orm

atio

n

Spr

edin

g asymp

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ce c

onta

ct a

rea

Adh

esio

n


8


Journal

ofEx

perim

entalB

iology






A B C E

D150 microm

01

0

10

20

30

02Capillary no

Adh

esio

nen

ergy

(10

ndash3 micro

J)

03


9


Journal

ofEx

perim

entalB

iology

































10


Journal

ofEx

perim

entalB

iology





























































11


Journal

ofEx

perim

entalB

iology
































12


Journal

ofEx

perim

entalB

iology





A

C

D

E


VCT

RT10 mm

B

40 microm


3


Journal

ofEx

perim

entalB

iology







Rel

ativ

e hu

mid

ity (

) 90

70

50

30

A

Date18 Aug 5 Sep

Rel

ativ

e hu

mid

ity (

)

Tem

pera

ture

(degC

)

Time (h)

B100

95

90

8 10 12 14 16 18 20

85

80

75

70

65

23

24

22

21

20

19

18

17

16

Abs

olut

e hu

mid

ity (g

mndash3

) C155

15

145

14

1358 10 12 14 16 18 20

Gly

copr

otei

n ar

eav

olum

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m2 microm

3 )

Leng

thg

lyco

prot

ein

volu

me

(microm

microm

3 )

D03

025

02

015

01

08

07

06

05

04

03

02

01

0


80 100

Rel

ativ

e w

ork

ofdr

ople

t ext

ensi

on

E250

200

150

100

50

Inse

ct re

tent

ion

time

(s)

F25

20

15

10

5


4


Journal

ofEx

perim

entalB

iology






A B

C D

Nocturnal

Neoscona crucifera

Forest edge

GABamide

Alanine

Glycine

Choline

N-Acetyltaurine

Putrescine

lsethionic acid

N-Acetylputrescine

Betaine

Taurine

Proline

Humidity

Araneus marmoreus

Forest interior

Verrucosa arenata

Open fieldsLow

Argiope aurantia

High

145

2

49

6

38

19

12

4

11

108

29

7

1025

15

6

6

11

21

6

9

4

18

86

15

14

12


5


Journal

ofEx

perim

entalB

iology







10 A B

C D

8

6

4

2

0P0 W0 W40

Conditions


Aromatic Aromatic

Aliphatic Aliphatic

120


50 0 200 150 100 50 0

110 100 90 80 120 110 100 90 80

ndashC=O ndashC=O

Stic

kine

ss (m

N)

Stic

kine

ss (micro

N)

Wwet W100 W100 P100

04

03

02

01

0


6


Journal

ofEx

perim

entalB

iology




7


Journal

ofEx

perim

entalB

iology






A B

C

Tetragnatha

30

0 s 01 s 1 s

50

70

90

7

4

3

2

2


Wor

k do

ne d

urin

g pe

elin

g (n

orm

aliz

ed J

)

70 80 90

Neoscona

Larinioides

Verrucosa

Argiope

Humidity

Viscosity

Hum

idD

ryFo

ragi

ng h

abita

t hum

idity

Bul

k di

ssip

atio

nasymp

resi

stan

ce to

def

orm

atio

n

Spr

edin

g asymp

surfa

ce c

onta

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rea

Adh

esio

n


8


Journal

ofEx

perim

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A B C E

D150 microm

01

0

10

20

30

02Capillary no

Adh

esio

nen

ergy

(10

ndash3 micro

J)

03


9


Journal

ofEx

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10


Journal

ofEx

perim

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11


Journal

ofEx

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iology
































12


Journal

ofEx

perim

entalB

iology







Rel

ativ

e hu

mid

ity (

) 90

70

50

30

A

Date18 Aug 5 Sep

Rel

ativ

e hu

mid

ity (

)

Tem

pera

ture

(degC

)

Time (h)

B100

95

90

8 10 12 14 16 18 20

85

80

75

70

65

23

24

22

21

20

19

18

17

16

Abs

olut

e hu

mid

ity (g

mndash3

) C155

15

145

14

1358 10 12 14 16 18 20

Gly

copr

otei

n ar

eav

olum

e (micro

m2 microm

3 )

Leng

thg

lyco

prot

ein

volu

me

(microm

microm

3 )

D03

025

02

015

01

08

07

06

05

04

03

02

01

0


80 100

Rel

ativ

e w

ork

ofdr

ople

t ext

ensi

on

E250

200

150

100

50

Inse

ct re

tent

ion

time

(s)

F25

20

15

10

5


4


Journal

ofEx

perim

entalB

iology






A B

C D

Nocturnal

Neoscona crucifera

Forest edge

GABamide

Alanine

Glycine

Choline

N-Acetyltaurine

Putrescine

lsethionic acid

N-Acetylputrescine

Betaine

Taurine

Proline

Humidity

Araneus marmoreus

Forest interior

Verrucosa arenata

Open fieldsLow

Argiope aurantia

High

145

2

49

6

38

19

12

4

11

108

29

7

1025

15

6

6

11

21

6

9

4

18

86

15

14

12


5


Journal

ofEx

perim

entalB

iology







10 A B

C D

8

6

4

2

0P0 W0 W40

Conditions


Aromatic Aromatic

Aliphatic Aliphatic

120


50 0 200 150 100 50 0

110 100 90 80 120 110 100 90 80

ndashC=O ndashC=O

Stic

kine

ss (m

N)

Stic

kine

ss (micro

N)

Wwet W100 W100 P100

04

03

02

01

0


6


Journal

ofEx

perim

entalB

iology




7


Journal

ofEx

perim

entalB

iology






A B

C

Tetragnatha

30

0 s 01 s 1 s

50

70

90

7

4

3

2

2


Wor

k do

ne d

urin

g pe

elin

g (n

orm

aliz

ed J

)

70 80 90

Neoscona

Larinioides

Verrucosa

Argiope

Humidity

Viscosity

Hum

idD

ryFo

ragi

ng h

abita

t hum

idity

Bul

k di

ssip

atio

nasymp

resi

stan

ce to

def

orm

atio

n

Spr

edin

g asymp

surfa

ce c

onta

ct a

rea

Adh

esio

n


8


Journal

ofEx

perim

entalB

iology






A B C E

D150 microm

01

0

10

20

30

02Capillary no

Adh

esio

nen

ergy

(10

ndash3 micro

J)

03


9


Journal

ofEx

perim

entalB

iology

































10


Journal

ofEx

perim

entalB

iology





























































11


Journal

ofEx

perim

entalB

iology
































12


Journal

ofEx

perim

entalB

iology






A B

C D

Nocturnal

Neoscona crucifera

Forest edge

GABamide

Alanine

Glycine

Choline

N-Acetyltaurine

Putrescine

lsethionic acid

N-Acetylputrescine

Betaine

Taurine

Proline

Humidity

Araneus marmoreus

Forest interior

Verrucosa arenata

Open fieldsLow

Argiope aurantia

High

145

2

49

6

38

19

12

4

11

108

29

7

1025

15

6

6

11

21

6

9

4

18

86

15

14

12


5


Journal

ofEx

perim

entalB

iology







10 A B

C D

8

6

4

2

0P0 W0 W40

Conditions


Aromatic Aromatic

Aliphatic Aliphatic

120


50 0 200 150 100 50 0

110 100 90 80 120 110 100 90 80

ndashC=O ndashC=O

Stic

kine

ss (m

N)

Stic

kine

ss (micro

N)

Wwet W100 W100 P100

04

03

02

01

0


6


Journal

ofEx

perim

entalB

iology




7


Journal

ofEx

perim

entalB

iology






A B

C

Tetragnatha

30

0 s 01 s 1 s

50

70

90

7

4

3

2

2


Wor

k do

ne d

urin

g pe

elin

g (n

orm

aliz

ed J

)

70 80 90

Neoscona

Larinioides

Verrucosa

Argiope

Humidity

Viscosity

Hum

idD

ryFo

ragi

ng h

abita

t hum

idity

Bul

k di

ssip

atio

nasymp

resi

stan

ce to

def

orm

atio

n

Spr

edin

g asymp

surfa

ce c

onta

ct a

rea

Adh

esio

n


8


Journal

ofEx

perim

entalB

iology






A B C E

D150 microm

01

0

10

20

30

02Capillary no

Adh

esio

nen

ergy

(10

ndash3 micro

J)

03


9


Journal

ofEx

perim

entalB

iology

































10


Journal

ofEx

perim

entalB

iology





























































11


Journal

ofEx

perim

entalB

iology
































12


Journal

ofEx

perim

entalB

iology







10 A B

C D

8

6

4

2

0P0 W0 W40

Conditions


Aromatic Aromatic

Aliphatic Aliphatic

120


50 0 200 150 100 50 0

110 100 90 80 120 110 100 90 80

ndashC=O ndashC=O

Stic

kine

ss (m

N)

Stic

kine

ss (micro

N)

Wwet W100 W100 P100

04

03

02

01

0


6


Journal

ofEx

perim

entalB

iology




7


Journal

ofEx

perim

entalB

iology






A B

C

Tetragnatha

30

0 s 01 s 1 s

50

70

90

7

4

3

2

2


Wor

k do

ne d

urin

g pe

elin

g (n

orm

aliz

ed J

)

70 80 90

Neoscona

Larinioides

Verrucosa

Argiope

Humidity

Viscosity

Hum

idD

ryFo

ragi

ng h

abita

t hum

idity

Bul

k di

ssip

atio

nasymp

resi

stan

ce to

def

orm

atio

n

Spr

edin

g asymp

surfa

ce c

onta

ct a

rea

Adh

esio

n


8


Journal

ofEx

perim

entalB

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A B C E

D150 microm

01

0

10

20

30

02Capillary no

Adh

esio

nen

ergy

(10

ndash3 micro

J)

03


9


Journal

ofEx

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10


Journal

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11


Journal

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12


Journal

ofEx

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entalB

iology




7


Journal

ofEx

perim

entalB

iology






A B

C

Tetragnatha

30

0 s 01 s 1 s

50

70

90

7

4

3

2

2


Wor

k do

ne d

urin

g pe

elin

g (n

orm

aliz

ed J

)

70 80 90

Neoscona

Larinioides

Verrucosa

Argiope

Humidity

Viscosity

Hum

idD

ryFo

ragi

ng h

abita

t hum

idity

Bul

k di

ssip

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stan

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orm

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Spr

edin

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surfa

ce c

onta

ct a

rea

Adh

esio

n


8


Journal

ofEx

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A B C E

D150 microm

01

0

10

20

30

02Capillary no

Adh

esio

nen

ergy

(10

ndash3 micro

J)

03


9


Journal

ofEx

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10


Journal

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11


Journal

ofEx

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entalB

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12


Journal

ofEx

perim

entalB

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A B

C

Tetragnatha

30

0 s 01 s 1 s

50

70

90

7

4

3

2

2


Wor

k do

ne d

urin

g pe

elin

g (n

orm

aliz

ed J

)

70 80 90

Neoscona

Larinioides

Verrucosa

Argiope

Humidity

Viscosity

Hum

idD

ryFo

ragi

ng h

abita

t hum

idity

Bul

k di

ssip

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Spr

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onta

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Adh

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8


Journal

ofEx

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A B C E

D150 microm

01

0

10

20

30

02Capillary no

Adh

esio

nen

ergy

(10

ndash3 micro

J)

03


9


Journal

ofEx

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Documents

Tuning orb spider glycoprotein glue performance to habitat ... · Spider Catalog, 2017). The order Araneae is composed of two suborders: Mesothelae, which have segmented abdomens