Tuning Orb Spider Glycoprotein Glue Performance to Habitat Humidity Brent D

Home , Araneus marmoreus, Argiope aurantia, Argiope trifasciata, Cribellum, Cyclosa turbinata, Cyrtophorinae

REVIEW Tuning orb spider glycoprotein glue performance to habitat humidity Brent D. Opell1,*, Dharamdeep Jain2, Ali Dhinojwala2 and Todd A. Blackledge3

ABSTRACT abdomen’s ventral surface; and Opisthothelae, which have Orb-weaving spiders use adhesive threads to delay the escape of unsegmented abdomens and posterior spinnerets (Platnick and insects from their webs until the spiders can locate and subdue the Gertsch, 1976). Opisthothelae contains two infraorders: insects. These viscous threads are spun as paired flagelliform axial Mygalomorphae, which includes tarantula and trapdoor spiders ’ fibers coated by a cylinder of solution derived from the aggregate whose cheliceral fangs move parallel to the body s sagittal plane; and glands. As low molecular mass compounds (LMMCs) in the aggregate Araneomorphae, which contains over 95% of all living spider solution attract atmospheric moisture, the enlarging cylinder becomes species whose fangs move more perpendicularly to the sagittal plane. unstable and divides into droplets. Within each droplet an adhesive Araneomorphae origin coincided with the appearance of a cribellum, glycoprotein core condenses. The plasticity and axial line extensibility a spinning plate formed of thousands of spigots that produces the of the glycoproteins are maintained by hygroscopic LMMCs. These nanofibers of a dry, fuzzy capture thread termed cribellate thread (see compounds cause droplet volume to track changes in humidity and Glossary). Although some araneomorphs continue to spin cribellate glycoprotein viscosity to vary approximately 1000-fold over the course threads (Opell, 2013), most no longer do so, constructing webs that of a day. Natural selection has tuned the performance of glycoprotein are not sticky or, like jumping spiders and wolf spiders, abandoning cores to the humidity of a species’ foraging environment by altering the web use in favor of other hunting tactics. The first orb webs contained composition of its LMMCs. Thus, species from low-humidity habits cribellate threads but 110 million years ago members of the have more hygroscopic threads than those from humid forests. superfamily Araneoidea replaced these with moist viscous capture However, at their respective foraging humidities, these species’ threads (see Glossary) (Peñalver et al., 2006). These viscous threads glycoproteins have remarkably similar viscosities, ensuring optimal are considered a key innovation (Bond and Opell, 1998), droplet adhesion by balancing glycoprotein adhesion and cohesion. contributing to the diversity of this clade, which contains 26% of Optimal viscosity is also essential for integrating the adhesion force of all spider species and comprises 17 families of orb-weaving spiders multiple droplets. As force is transferred to a thread’s support line, and their descendants that spin webs with divergent architectures extending droplets draw it into a parabolic configuration, implementing (Blackledge et al., 2009a,b; Dimitrov et al., 2016; Hormiga and a suspension bridge mechanism that sums the adhesive force Griswold, 2014). generated over the thread span. Thus, viscous capture threads Organisms employ adhesive secretions for a variety of other extend an orb spider’s phenotype as a highly integrated complex of functions. For example, Polychaeta annelids construct protective ̌ large proteins and small molecules that function as a self-assembling, tubes from cemented sand particles (Pavlovic et al., 2014), barnacles highly tuned, environmentally responsive, adhesive biomaterial. cement their cases to rocks and mussels attach themselves by byssal Understanding the synergistic role of chemistry and design in spider threads to the substrate to avoid being swept away by currents adhesives, particularly the ability to stick in wet conditions, provides (Kamino, 2010; Waite, 2017). Like most commercial adhesives, insight in designing synthetic adhesives for biomedical applications. bioadhesives typically have an initial low-viscosity phase, during which they establish surface contact, followed by a phase of KEY WORDS: Adhesive, Biomaterial, Hygroscopic, Prey capture, increased stiffness, which allows them to resist the crack Self-assembling propagation that leads to failure (Gent, 1996). English ivy clings to tree trunks by secreting a low-viscosity adhesive solution that Introduction – spider diversity and the role of prey capture spreads before water evaporates, hardening it into a matrix (Huang thread et al., 2016). However, the challenge is much greater for aquatic Evolution in silk use has played a crucial role in the success of the animals (Stewart et al., 2011). Barnacles and mussels solve the diverse, over 47,000-species-strong arachnid order Araneae to which problem by secreting adhesives that are subsequently enzymatically spiders belong (Vollrath, 2005; Vollrath and Selden, 2007; World hardened (Dickinson et al., 2009; Naldrett, 1993; So et al., 2016; Spider Catalog, 2017). The order Araneae is composed of two Waite, 2017). By contrast, the glycoprotein (see Glossary) glue of suborders: Mesothelae, which have segmented abdomens like an orb-weaving spider’s viscous threads remains hydrated and scorpions and spinnerets that extend from the middle of their pliable in air because it is contained in tiny aquatic spheres (Fig. 1D,E) (Edmonds and Vollrath, 1992; Tillinghast et al., 1993; Townley et al., 1991). This ensures that their glycoprotein adhesive retains its 1Department of Biological Sciences, Virginia Tech, Blacksburg, VA 24061, USA. viscoelasticity for effective adhesion (Sahni et al., 2010). 2Department of Polymer Science, Integrated Bioscience Program, The University of Orb-weaving spiders integrate silk produced from four distinct Akron, Akron, OH 44325, USA. 3Department of Biology, Integrated Bioscience Program, The University of Akron, Akron, OH 44325, USA. silk glands into a highly effective prey capture web (Fig. 1A). Attached by pyriform gland (see Glossary) secretions (Sahni et al., *Author for correspondence ([email protected]) 2012a; Wolff et al., 2015), non-adhesive radial and frame threads B.D.O., 0000-0002-1830-0752; A.D., 0000-0002-3935-7467; T.A.B., 0000-0002- produced by major ampullate glands (see Glossary) absorb and

8166-5981 dissipate the kinetic energy of an insect’s impact (Sensenig et al., Journal of Experimental Biology

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Glossary Young’s modulus Aciniform glands Also referred to as elastic modulus, describes the stiffness of a material, Spinning glands that produce large amounts of silk used to wrap and and is expressed as the energy per cross-sectional area required to immobilize prey. extend a material. Lower values denoting more easily stretched material. Aggregate gland Young’s modulus is determined as the slope of the linear region of a One of two spinning glands that open at the tips of adjacent spigots on material’s stress–strain curve. each posterior lateral spinneret (Fig. 1B) and together coat a flagelliform fiber with a solution of inorganic salts and organic molecules, which are reconfigured to form the outer lipid layer, aqueous layer, glycoprotein core and central granule of a viscous capture thread droplet. 2012), while spirally arrayed, adhesive prey capture threads Aqueous layer produced from flagelliform and aggregate glands (see Glossary) ’ The material that covers a viscous thread s axial lines and adhesive retain the insect (Sahni et al., 2013) until the spider can locate, run to glycoprotein cores. This solution contains the low molecular mass compounds and inorganic salts that confer thread hygroscopicity and and begin to subdue it. Material invested in non-adhesive threads condition and solvate the glycoproteins in the core of a viscous droplet. influences the size and velocity of insects that a web can stop This layer is composed of aggregate gland material that remains after the (Sensenig et al., 2012), and material invested in capture thread glycoprotein cores of droplets are formed. affects the time an insect is trapped (Opell et al., 2017). A retention Axial lines or axial fiber time (see Glossary) difference of even a few seconds can be the One of two protein strands that is spun from a flagelliform gland spigot on difference between a prey being captured or lost (Eberhard, 1989). each posterior lateral spinneret and serves as one of the central support lines of a viscous capture thread. Orb-weaving spiders are not unique in relying on extended Cribellate thread phenotypes (see Glossary) for important functions (Dawkins, Plesiomorphic type of dry prey capture thread comprising an outer layer 1982), nor are they the only animals that use these products for of thousands of nanofibrils that surround larger supporting fibers. prey capture. For example, parchment worms and caddisfly larvae Extended phenotype employ nets to filter organic material from the water (Flood and A physical product or construction of an animal that is genetically Fiala-Médioni, 1982; Mackay and Wiggins, 1979). Like other determined, affects its fitness and, therefore, can be shaped by natural extended phenotypes (see Glossary), orb spider threads and webs selection. Flagelliform gland exhibit physical and architectural plasticity (Blamires, 2010; A spinning gland that opens at the tip of a spigot found on each of the Blamires et al., 2014, 2016, 2017; Crews and Opell, 2006; posterior lateral spinnerets (Fig. 1B) and contributes one of the two Herberstein and Tso, 2011; Scharf et al., 2011; Townley et al., supporting axial lines of a viscous capture thread. 2006; Tso et al., 2007; Wu et al., 2013). However, viscous threads Foraging humidity are unusual in that, after being spun, they continue to exhibit ’ Humidity during the longest portion of an orb weaver s feeding period. plasticity as they respond to environmental conditions, most notably Except for nocturnal species, this corresponds to the times of lower humidity that occur from mid-morning to late afternoon. relative humidity (RH) (see Glossary) (Agnarsson et al., 2009; Glycoprotein Opell et al., 2011a, 2013; Sahni et al., 2011; Stellwagen et al., A polypeptide chain with attached carbohydrate groups. These are 2015a, 2014). considered the primary adhesives of viscous prey capture threads. Temperature and ultraviolet light influence viscous thread Low molecular mass compounds (LMMCs) properties and performance (Stellwagen et al., 2015b, 2016, Small organic and inorganic molecules that are present in aggregate 2014), although humidity has the greatest and most universal gland secretions and remain in a viscous thread’s aqueous layer. These are largely responsible for a viscous thread’s hygroscopicity and serve to effect. As RH decreases during daylight hours, temperature solvate and condition its glycoprotein adhesive. increases, mediating the decrease in absolute humidity and Major ampullate gland reducing glycoprotein viscosity. However, species experience the Spinning gland that produces non-adhesive threads that form an orb impact of humidity differently. Orb weavers that live in exposed, web’s attachment, frame and radial lines (Fig. 1A). weedy vegetation experience greater daily oscillations in humidity Plateau–Rayleigh instability than those whose webs are anchored in vegetation that provides The phenomenon by which the surface tension of the liquid in a thin shade and helps maintain humidity (cf. Fig. 2A,B). Although the stream or, in the case of viscous threads, a thin coating is lowered by the formation of small drops that minimize surface area. first study of the effect of humidity on viscous thread adhesion was Pyriform glands published over 30 years ago (Strohmenger and Nentwig, 1987), Spinning glands that open in a cluster of spigots on a spider’s anterior renewed interest in this topic is revealing details about the impact of lateral spinnerets and produce a dense, zig-zag array of fibers that attach humidity on this complex and highly integrated natural adhesive major ampullate threads to a substrate. system. Relative humidity (RH) Water vapor pressure expressed as a percentage of maximum water vapor pressure at a given temperature and described by the formula: Viscous capture thread production RH=(actual vapor pressure/saturated vapor pressure)×100%. Understanding the response of viscous threads to environmental Retention time humidity is key to understanding both the function and evolution of The time an insect is retained by an orb web’s prey capture threads this unique adhesive system. A viscous thread is a compound, self- before it can escape. assembling adhesive produced by two aggregate gland spigots Suspension bridge mechanism flanking a conical flagelliform gland (see Glossary) spigot (Fig. 1B) Viscous thread’s ability to sum the adhesive forces generated by multiple ’ droplets as they extend, transferring force to the thread’s flagelliform on each of a spider s paired posterior lateral spinnerets, a total of six axial lines, which have assumed a parabolic configuration (Fig. 7). spigots contributing to each thread (Coddington, 1989; Park and Viscous capture thread Moon, 2014; Peters, 1955). As an axial line (see Glossary) emerges The wet prey capture thread of araneoid spiders that features glycoprotein from the flagelliform spigot’s tip, it is coated with aggregate gland adhesive covered by a hygroscopic aqueous layer (Fig. 1A,D). solution that contains glycoprotein and small hygroscopic

molecules (Townley and Tillinghast, 2013). The coated axial Journal of Experimental Biology

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A B Fig. 1. Viscous capture thread production and composition. (A) A female Argiope aurantia spins a viscous capture thread (VCT) prior to attaching it to a major ampullate radial thread (RT). (B) Scanning electron microscope image of the spinning spigots on one posterior lateral spinneret that are responsible for producing a viscous capture thread. AG, aggregate gland spigots; FL, µ 40 m flagelliform gland spigot. (C) An Argiope trifasciata thread showing droplets forming from the aggregate material cylinder. (D) The same thread less than 30 s later after droplets have formed. (E) A Neoscona crucifera droplet that has been flattened against a glass coverslip at 90% relative humidity to show its glycoprotein core attached to flagelliform VCT axial fibers and surrounded by aqueous material. Panel B adapted from Blackledge et al. (2009a).

RT 10 mm C E

200 µm 30 µm

fibers (see Glossary) from each of the two spinnerets merge to form whereas the granule is more easily seen with transmitted light, a single cylindrical thread, after which Plateau–Rayleigh instability where it appears as a cylinder or toroid within the core (Opell and (see Glossary) causes the aggregate material to quickly form a series Hendricks, 2010). Consequently, in some older literature the of evenly spaced droplets that exhibit a bead on a string (BOAS) granule is assumed to be responsible for thread adhesion. It is not morphology (Fig. 1C,D) (Edmonds and Vollrath, 1992; Mead- known if the granule is simply a region of the glycoprotein that has Hunter et al., 2012; Roe, 1975). Environmental humidity affects the become associated with flagelliform fibers or a distinct protein or size of the droplets that form through its impact on the viscosity of proteins. Although droplets resist being moved along the axial the aggregate material (Edmonds and Vollrath, 1992; Sahni et al., fibers, they are not permanently bonded and can slide (Opell et al., 2012b). Studies of viscous thread analogs and droplet formation in 2011a, 2013). thin films show that the velocity of thread production and the size and Despite the large percentage of water in a droplet, the adhesion of shape of nozzle apertures affect droplet spacing (Sadeghpour et al., its glycoprotein is several orders of magnitude greater than the 2017; Sahni et al., 2012b), principles worth examining in viscous capillary adhesion of its aqueous layer (Sahni et al., 2010). Only one thread spinning. At the center of each droplet a glycoprotein core thread glycoprotein, aggregate spider glue 2 or ASG2 has been coalesces (Fig. 1E) (Vollrath and Edmonds, 1989). Although this is characterized (Choresh et al., 2009; Collin et al., 2016; the only droplet region where protein can be visualized under light Vasanthavada et al., 2012), with ASG1 subsequently being microscopy, proteins are also found in the remaining aqueous associated with mucin proteins that bind chitin to cells (Collin material, which covers both the thread’s supporting axial fibers and et al., 2016). Collin et al. (2016) showed that ASG2 is a member of its glycoprotein cores (Amarpuri et al., 2015a). the spidroin gene family and suggested that, consistent with spidroin nomenclature, it be named aggregate spidroin 1 (AgSp1). Spidroins Viscous capture thread structure and composition are a class of scleroproteins that includes major ampullate and Four droplet regions have been identified: (1) a thin outer lipid coat, flagelliform fibers (Ayoub et al., 2007; Garb et al., 2010, 2007; first identified by Hans Peters (Peters, 1995) and seen as a ‘skin’ in Gatesy et al., 2001). However, the presence of AgSp1 proteins in scanning electron microscope images of desiccated droplets (Opell glue droplets has not been confirmed and we do not know whether and Hendricks, 2009) but poorly studied; (2) the aqueous layer (see AgSp1 is the only glycoprotein gene or if this type of protein is the Glossary) containing proteins and the small molecules that are only adhesive in a droplet. The challenge of adhering to an insect’s described in the following section; (3) a distinct glycoprotein core; waxy epicuticle is great, and our understanding of AgSp1’s mode of and (4) a granule in the core’s center, which is thought to anchor the adhesion is poor relative to that of other bioadhesives, such as core to the thread’s flagelliform fibers (Opell and Hendricks, 2010). mussel glue (Forooshani and Lee, 2017). Although glycoproteins Both the glycoprotein core and its granule are most clearly seen are known to be adhesives (e.g. von der Mark and Sorokin, 2002; when a droplet has been flattened on a microscope slide or coverslip. Xu and Mosher, 2011), until information about possible

Epi-illumination more clearly reveals the glycoprotein core, post-translational modifications of AgSp1 proteins and their Journal of Experimental Biology

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A 0.3 D 0.8 ) ) 3

90 3 m

m 0.7 µ µ / 2 m/ µ 70 m 0.25 0.6 µ

0.5 50 0.2 0.4

Relative humidity (%) 30 0.3 18 Aug. 5 Sep. Date 0.15 0.2

100 B 24 0.1 Glycoprotein area/volume ( 23 Length/glycoprotein volume ( 95 0.1 0 22 90 21 250 E 85 20 200 80 19 150

75 (°C) Temperature 18 100 Relative humidity (%) Relative work of 70 17 droplet extension 50 65 16 8 101214161820 F 15.5 C 25 ) –3

15 20

14.5 15

14 10 Insect retention time (s) Absolute humidity (g m 13.5 5 8 101214161820 0 20 40 60 80 100 Time (h) Relative humidity (%)

Fig. 2. Daily changes in environmental humidity and its effect on viscous thread properties and insect retention time. (A) Daily changes in relative humidity (RH) in the exposed weedy vegetation habitat of Argiope aurantia during 2011. (B) RH and temperature in the forest edge habitat of Araneus marmoreus from 15 August to 15 October 2016. (C) Mean absolute humidity in this A. marmoreus habitat. (D) Volume-specific glycoprotein flattened area (solid circles) and extension (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 five humidities. (F) Active struggle time required by a housefly to escape from three capture thread strands, showing the association of viscous droplet and thread features with insect retention time. Images to the right of panels D–F illustrate the properties that are plotted. Error bars are ±1 s.e. Panels D–Fare observations made at 23°C. Panel A is adapted from Opell et al. (2013) and panels B–F from Opell et al. (2017). three-dimensional structure is available, it will be difficult to 2012, 2006; Townley and Tillinghast, 2013; Vollrath et al., 1990). − + − + − 2+ determine their mode of adhesion. Inorganic LMMCs include H2PO4 ,K ,NO3 ,Na ,Cl and Ca moieties (Anderson and Tillinghast, 1980; Townley and Viscous thread response to humidity in a spider’s Tillinghast, 2013; Townley et al., 2006; Vollrath et al., 1990). environment The LMMCs are hypothesized to have evolved in part from Viscous glue droplets contain abundant amounts of water-soluble neurotransmitters (Edmonds and Vollrath, 1992), but are now organic and inorganic compounds that are hygroscopic in nature. distributed throughout the aqueous material where they function to These are often termed low molecular mass compounds take up water from the environment and interact with glycoproteins (LMMCs) (see Glossary). Most are organic and only 10–20% to render the glue functional in different humidity conditions are inorganic compounds (Townley and Tillinghast, 2013). (Amarpuri et al., 2015b; Opell et al., 2013; Sahni et al., 2011, 2014; Organic LMMCs are small polar aliphatic compounds (mostly Townley and Tillinghast, 2013). Individual LMMCs differ widely amine and sulphate based), such as alanine, choline, betaine, in hygroscopic response. Compounds such as choline and N- proline, glycine, taurine, GABamide, putrescine, N-acetyltaurine, acetyltaurine are hygroscopic over a range of humidity conditions; N-acetylputrescine and isethionic acid (Fig. 3) (Anderson and GABamide, N-acetylputrescine and isethionic acid start adsorbing

Tillinghast, 1980; Tillinghast et al., 1987; Townley et al., 1991, at approximately 55% RH, whereas glycine, potassium nitrate and Journal of Experimental Biology

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Fig. 3. Diversity of organic low molecular ABNeoscona crucifera Araneus marmoreus mass compounds (LMMCs) in viscid glues of 2% orb web spiders. (A–D) Relative compositions of 5% diverse organic LMMCs (color coded as depicted 12% 14% 6% in key) present in the glues of orb webs belonging to Neoscona crucifera, Araneus marmoreus, 14% 9% Verrucosa arenata and Argiope aurantia,each inhabiting a habitat with a different foraging 4% 21% GABamide 49% humidity (see Glossary). Not only do the 15% Alanine percentage compositions of LMMCs such as 18% GABamide and choline differ among species but 11% Glycine some LMMCs are restricted to certain species. 6% 8% For example, taurine is found only in A. aurantia, 6% Choline isethionic acid is found only in A. marmoreus and A. aurantia, and betaine is present in all species N-Acetyltaurine Nocturnal Forest edge but A. marmoreus. These differences are Putrescine explained by many factors that probably include the hygroscopic strength of the LMMCs, their CDVerrucosa arenata Argiope aurantia lsethionic acid metabolic costs, competition for these 6% compounds across metabolic processes and 8% N-Acetylputrescine phylogenetic relationship among the species 6% 10% represented. The effect on each species’ unique Betaine 29% mix of LMMCs on droplet hygroscopicity is shown in Fig. 5C and on thread adhesion at different 11% Taurine 15% 38% humidities in Fig. 6A. Proline 4% 7% 12% 25% 10% Humidity 19% High

Forest interior Open fields Low potassium dihydrogen phosphate show less than 3% water uptake enhanced adhesion. When LMMCs are washed off, the viscid glue by mass even at high humidity conditions (Townley et al., 1991; is irresponsive to humidity (Fig. 4D) and the glycoproteins become Vollrath et al., 1990). LMMCs differ in their types and rigid, corresponding to the collapse of the glue at a macro level compositions across orb-weaving species living in habitats with (Sahni et al., 2014). Altering LMMCs composition provides a different humidity levels (Fig. 3). However, it is important to note mechanism by which natural selection can optimize viscous thread that even among individuals of the same species, LMMCs performance to the humidity in a species’ environment. composition differs and is presumed to be affected by a spider’s Viscous droplet volume responds dramatically to changes in genetics and diet (Higgins et al., 2001). humidity (Fig. 5A) (Opell et al., 2011a, 2013). However, as we will The primary function of the LMMCs is to solvate and soften explain, the degree of droplet hygroscopicity differs among species glycoproteins to enhance adhesion. The LMMCs interact with the and is related to the humidity of a species’ habitat. Glycoprotein glycoproteins to make viscid glue functionally responsive to volume also responds to humidity (Fig. 5C), documenting that, after humidity in the environment. Pristine thread droplets swell as RH atmospheric water enters a droplet’s aqueous layer, some of it is increases, whereas removal of the hygroscopic compounds by absorbed by the glycoprotein core. This results in an increase in washing threads with water leads to the collapse of the glue structure droplet extensibility as humidity increases (Fig. 5B). Even after and renders it incapable of subsequently taking up more than extension is adjusted for glycoprotein volume, this response differs 10–20% water even at high humidity. After this collapse, it becomes among species (Fig. 5D,E). Compared with the lower hygroscopic impossible to reintroduce LMMCs back into the washed glue to droplets of species such as Neoscona crucifera and Verrucosa recover adhesion, and at 100% RH washed threads lose two orders arenata that occupy humid environments, the more hygroscopic of magnitude of adhesion compared with pristine threads droplets of Argiope aurantia and Larinioides cornutus do not extend (Fig. 4A,B). In all conditions (0%, 40%, 100% RH or wet), as far at higher humidities before releasing because their glycoprotein washed glue droplets fail to make intimate contact and do not adhere more easily becomes over lubricated, dropping in viscosity and more to the surface (Sahni et al., 2014). Various solid-state nuclear easily releases from a surface (Fig. 5D) (Opell et al., 2013; Sahni magnetic resonance (NMR) spectroscopy techniques have shown et al., 2011). Thus, the viscosity of A. aurantia glycoprotein at 55% that the glycoproteins soften and become humidity responsive in the RH is similar to that of N. crucifera at 90% RH (Fig. 5D,E). Although presence of LMMCs. Cross-polarization magic-angle spinning the greater hygroscopicity of A. aurantia threads might appear to be a (CPMAS) NMR is sensitive to rigid molecules and demonstrates deficiency, it is, in fact, an adaptation to remaining hydrated during that the rigidity of glycoproteins in pristine glue decreases as the late morning and afternoon hours when humidity is low (Fig. 2A). humidity is increased from 0% RH to 100% RH (indicated by the The level of humidity at which adhesion of viscid glues reaches a decrease in intensity of the spectrum in Fig. 4C). This directly maximum in different spider species corresponds to their foraging correlates with macro-level observations of glue getting softer as habitats (Fig. 6A). Maximum adhesion occurs when the viscosity of humidity rises, resulting in intimate contact with surfaces and the glue is such that the contribution of two factors is optimized: Journal of Experimental Biology

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ABFig. 4. Interaction of low molecular mass 10 0.4 compounds (LMMCs) and glycoproteins in adhesion of viscid threads. 8 (A,B) Adhesion forces for pristine (P) and 0.3 washed (W, obtained after removal of N) µ 6 LMMCs) capture silk threads of Larinioides 0.2 cornutus tested on glass substrates under different conditions [P0, W0: desiccated; 4 P100, W100: 100% relative humidity (RH); Stickiness ( 0.1 Stickiness (mN) W40: 40% RH; Wwet: externally wetted]. 2 (C,D) Cross-polarization magic-angle spinning solid-state nuclear magnetic 0 0 resonance measurements for pristine P0 W0 W40 Wwet W100 W100 P100 (C) and washed (D) capture silk threads of Conditions L. cornutus, recorded at 0% RH (blue), 35% RH (green) and 100% RH (red). Adapted CD–C=O –C=O and reprinted with permission from Sahni, Glycoprotein Glycoprotein V., Miyoshi, T., Chen, K., Jain, D., Blamires, Aliphatic Aliphatic S. J., Blackledge, T. A. and Dhinojwala, A. (2014). Direct solvation of glycoproteins by salts in spider silk glues enhances adhesion and helps to explain the evolution of modern spider orb webs. 120 110 100 90 80 120 110 100 90 80 Biomacromolecules 15, 1225-1232. Copyright 2014 American Chemical Aromatic Aromatic Society.

200 150 100 50 0 200 150 100 50 0 13C chemical shift (ppm) surface interactions (substrate–glue interaction energy and an insect’s exoskeleton (Bott et al., 2017). Although versatile, the spreading area); and bulk dissipation (rate of peeling and adhesion of this thread is limited by the stiffness of its internal viscosity) (Amarpuri et al., 2015b). As RH increases, spreading supporting fibers. Its adhesion does not increase as increasing of the droplets improves as bulk dissipation decreases (Fig. 6B). At lengths of thread contact a surface, indicating that, after the adhesion low humidity, droplets are stiff and do not spread efficiently. As of terminal thread regions fails, crack propagation ensues, humidity increases, droplets spread and resist peeling as the preventing additional adhesion being recruited from more central glycoprotein extends, leading to generation of high adhesive thread regions (Opell and Schwend, 2008). forces. At high humidity, droplets coalesce to form a sheet of glue In contrast, viscous thread adhesion increases as the thread that spreads completely but breaks easily. These changes in contact length increases (Opell and Hendricks, 2007, 2009). The behavior represent a remarkable 1000-fold variation in glue pliable adhesive droplets of viscous threads combine with the viscosity, but adhesion is maximized in a relatively narrow range thread’s extensible flagelliform support lines (Blackledge and of viscosity that optimizes spreading and bulk contributions Hayashi, 2006) to create a dynamic adhesive system that assumes (Fig. 6C). Remarkably, this optimal viscosity is achieved at very the configuration of a ‘suspension bridge’ as it sums the adhesive different humidities in different species that closely matches where forces of multiple droplets (Fig. 7). Moreover, as force is applied to each forages (Fig. 6A). Thus, the diverse mixture of LMMCs a thread, the extension of its droplets and flagelliform lines (Fig. 3) adapts species to a range of habitat humidities (Amarpuri combines to dissipate the energy of a struggling prey (Piorkowski et al., 2015b; Opell et al., 2015, 2013). In the next section, we and Blackledge, 2017; Sahni et al., 2011). Thus, there are two ways explain why maintaining glycoprotein extensibility plays an to characterize viscous thread adhesion: the force required to pull a important role in thread adhesion. thread from a surface (e.g. Opell and Hendricks, 2007, 2009); and the work of adhesion required to bring a thread to the point of pull- Summing the adhesive forces of individual droplets off (e.g. Sahni et al., 2011). In the milliseconds after an insect strikes a web, a viscous capture The thread’s hygroscopic aqueous layer also makes an essential thread’s glycoprotein cores must spread immediately to establish contribution to the suspension bridge mechanism (see Glossary) by adhesion and then, as the insect struggles to escape, instantly resist ensuring that flagelliform fibers remain hydrated and extensible. shifting forces that threaten to pull threads from the insect’s body When threads were stretched experimentally to reduce axial fiber and wings. If the axial lines and droplets were rigid, force applied to extensibility, but the number of contributing droplets was a thread would cause the terminal droplets to release and initiate maintained by contacting longer thread lengths, the force required serial droplet pull-off that would quickly lead to thread release. to pull a thread from a surface decreased (Opell et al., 2008). Compared with cribellate thread, the plesiomorphic, dry prey Flagelliform fiber extension is also crucial for a thread’s ability to capture threads spun by araneoid ancestors (Garrison et al., 2016), dissipate the energy of a struggling insect (Sahni et al., 2011), viscous thread is more effective in this regard. Cribellate threads are contributing more than twice the work of adhesion as combined formed of several thousand dry protein nanofibers arrayed around droplet extensions (Piorkowski and Blackledge, 2017). support lines and can adhere by van der Waals forces, capillary Because viscous threads rely on the extensibility of both attachment, snagging on insect setae (Joel et al., 2015; Opell, 2013) flagelliform fibers and the glycoprotein cores of droplets the and can even embed their nanofibrils in the waxy outer epicuticle of performance of these two components must have evolved in a Journal of Experimental Biology

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Fig. 5. The effect of humidity on viscous thread droplet volume, glycoprotein volume and droplet extensibility at 23°C. (A) The same Argiope aurantia droplet imaged at three relative humidities. (B) The impact of relative humidity on the extensibility of A. aurantia droplets. (C) Increases in droplet and glycoprotein volumes of five orb weavers that occupy different habitats. (D) The extension of A. aurantia droplets at different humidities relative to a droplet’s glycoprotein volume. (E) The extension of N. crucifera droplets at different humidities relative to a droplet’s glycoprotein volume. Above 55% relative humidity (RH), A. aurantia glycoprotein becomes over lubricated, causing it to pull from a surface before its full extension is expressed. In contrast, N. crucifera droplets attract less moisture, causing glycoprotein viscosity to decrease and extension to increase, but never absorb enough moisture to become over lubricated. Diagrams below panels D and E depict this decrease in a glycoprotein viscosity with increasing humidity as seen in a droplet’s contact footprint that is circled on the left of each series. Error bars are ±1 s.e. Adapted from or constructed from data in Opell et al. (2013) and B.D.O., unpublished. complementary fashion. If glycoprotein is too stiff relative to a great, there will be little resistance and the axial line will bow thread’s flagelliform fibers, the outer droplets of a contacting strand acutely, with little work being done and little adhesive force being will release before inner droplets have extended and contributed summed. This is borne out by a comparison of the Young’s their adhesive forces. If, by contrast, glycoprotein extensibility is too modulus (see Glossary) of three species’ flagelliform fibers and Journal of Experimental Biology

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B A 0 s 0.1 s 1 s

30% Humid Tetragnatha 7ϫ 50%

70%

4ϫ Neoscona

90% C Humidity Larinioides 3ϫ Foraging habitat humidity Verrucosa 2ϫ Spreding Work done during peeling (normalized, J) Work Bulk dissipation ≈ surface contact area ≈ resistance to deformation Argiope

2ϫ Dry 30 40 50 60 70 80 90 Adhesion Relative humidity (%) Viscosity

Fig. 6. Tuning viscous thread to habitat humidity. (A) Maximum adhesion response as a function of humidity for capture silk threads belonging to species occupying 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 µm. (C) Diagram showing how glycoprotein spreading (red) and bulk dissipation or viscosity (green) trends must be balanced to produce an optimized adhesion response. Adapted and reprinted with permission from Amarpuri, G., Zhang, C., Diaz, C., Opell, B. D., Blackledge, T. A. and Dhinojwala, A. (2015). Spiders tune glue viscosity to maximize adhesion. ASC Nano. 9, 11472-11478. Copyright 2015 American Chemical Society. glycoproteins. Young’s modulus (E) is a measure of a material’s necessary for spider physiology and are in short supply, being stiffness, with smaller values indicating a material that is more obtained only from insect prey and ingested threads (Higgins and easily extended. When compared at 50% RH, flagelliform E ranged Rankin, 1999; Townley and Tillinghast, 2013; Townley et al., 2006). from 0.009 to 0.0300 GPa and glycoprotein E from 0.00003 to As we gain a greater understanding of viscous thread 0.0014 GPa, with flagelliform E being 21, 52 and 290 times greater hygroscopicity and fine-scale, humidity-mediated changes in than glycoprotein E for the three species (B.D.O., M. E. Clouse and viscous droplets, it is important to determine how these features S. F. Andrews, unpublished; Sensenig et al., 2010). impact prey retention time because this is ultimately how natural selection must tune thread performance to the humidity of a species’ Physiological and ecological impact of humidity environment. However, assessing prey retention, particularly in As the studies of Tillinghast, Townley, Vollrath and their colleagues vertically oriented orb webs like most of those that have been have shown (Edmonds and Vollrath, 1992; Townley et al., 1991, studied, is challenging. Retention is affected by many factors, 2012, 2006; Townley and Tillinghast, 2013; Vollrath et al., 1990; including the mass of an insect and its impact velocity, the number Vollrath and Tillinghast, 1991), environmental humidity plays a of capture threads that it strikes, the texture of the insect’s body crucial role in the function of an orb web from the time that it is region that contacts a thread, the region of the web a prey strikes and constructed until it is taken down and its silk ingested. High whether, after struggling free from these threads, the insect tumbles humidity during the later evening and early morning hours when into other capture threads (Blackledge and Zevenbergen, 2006; most orb webs are constructed affects the self-assembly of the glue Opell and Schwend, 2007; Sensenig et al., 2013; Zschokke and droplets of viscous capture threads. Changes in humidity over the Nakata, 2015). course of a day (Fig. 2A–C) affect the web’s ability to both withstand To make humidity the focal variable, an anesthetized housefly prey impact (Boutry and Blackledge, 2013) and retain intercepted was placed wings downward across three, equally spaced, prey (Opell et al., 2017). Finally, when ingested the fully hydrated horizontal capture thread strands from the large orb weaver glue droplets supply a spider with both water and recyclable Araneus marmoreus (Fig. 2F) and its escape captured in a video nutrients (Edmonds and Vollrath, 1992; Townley and Tillinghast, recording (Opell et al., 2017). The humidity maximizing retention

1988). In fact, some important LMMCs like choline are also time of the flies was predicted to be the humidity at which both the Journal of Experimental Biology

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humidity similar to viscid silk. The unique natural designs of both cribellate and viscous prey capture threads have inspired researchers to develop similarly structured materials for a variety of applications, including adhesives, water collectors and solid–liquid hybrid materials (Bai et al., 2012; Chen and Zheng, 2014; Elettro et al., 2016; Sahni et al., 2012b; Song et al., 2014; Tian et al., 2011). In one of the first attempts, synthetic adhesive BOAS microthreads were fabricated by drawing a synthetic nylon thread through a pool of polydimethylsiloxane (PDMS) polymer (Sahni et al., 2012b). The process created a cylindrical coating that formed smaller droplets due to Plateau–Rayleigh instability and these threads were sticky when tested on a glass substrate (Fig. 8). The spacing and diameter of these synthetic thread droplets were varied by changing the capillary number (Ca=velocity×viscosity/surface tension), which depends on drawing velocity, PDMS viscosity and surface tension (Fig. 8A–C). A higher capillary number (higher velocity, higher viscosity and lower surface tension) produced larger and more widely spaced droplets (Fig. 8C), which exhibited greater adhesion (Fig. 8E). The study presented a simple and effective manner of creating BOAS adhesive mimics of viscous threads (Fig. 8D) and also helped in Fig. 7. A single Verrucosa arenata capture thread being pulled from a testing the fundamental principles behind the adhesion of viscid silk 2 mm wide contact plate. Adhesive forces from the thread’s progressively by using synthetic mimics (Sahni et al., 2012b). This successful extending droplets are summed by being collectively transferred to the strategy can also be used to generate humidity-responsive adhesives. deflected axial line. In the top frame, a droplet near the strand’s center has For example, droplets can be laden with mixtures of LMMCs released from the plate, introducing an instability that will initiate adhesive mimicking natural compositions (Fig. 3) incorporated within failure. polymer matrices to generate viscous thread to synthesize humidity-sensitive adhesives. These synthetic adhesive structures surface area and extensibility of the glycoprotein were greatest can then be used in applications such as a bandages or adhesive tapes (Fig. 2D). This occurred at 72% RH; the same level at which the where adhesion is crucial in the presence of water. energy estimated to bring a 4 mm span of capture thread to the initiation of pull-off was greatest and thus, most difficult for a prey ABC E to achieve (Fig. 2E). This humidity is also similar to the afternoon 30 J) µ humidity at the forest edge where A. marmoreus lives (Fig. 2B). At 72% RH, actively struggling flies were retained 11 s longer than at –3 20 10 either 37% or 55% RH (Fig. 2F). This additional time is ϫ 10 ecologically significant because it provides a spider more time to Adhesion locate and reach an insect and to begin wrapping it with silk from numerous aciniform gland (see Glossary) spigots on the posterior energy ( 0 median and posterior lateral spinnerets (Coddington, 1989; 0.1 0.2 0.3 Tremblay et al., 2015) before the prey can escape the web. 150 µm Capillary no. Greater retention times also relate directly to the size of insects that a web can retain. For large orb weavers such as A. marmoreus,it D is postulated that these large, rare prey are more profitable and comprise the greatest proportion of a spider’s total food intake (Blackledge, 2011; Venner and Casas, 2005) but see Eberhard (Eberhard, 2013) for challenges to this hypothesis. Thus, there is solid evidence that longer prey retention time selects for changes in the composition of a viscous thread’s hygroscopic compounds that tune thread performance to the humidity of a species’ habitat. These findings are the first step in ascribing fitness values to the performance characteristics of viscous threads. As data for other species are added, it should be possible to rank the relative Fig. 8. Synthetic adhesive threads and their performance. (A–C) Adhesive contributions of glycoprotein surface area, viscosity and extension polydimethylsiloxane (PDMS) microthreads with differences in droplet spacing to prey retention time. and diameter resulting from differences in the velocity with which nylon threads were drawn through a PDMS solution. (D) Image showing the formation of a Synthetic viscous threads as models for adhesives suspension bridge when a synthetic microthread is pulled from a glass Humidity poses serious problems to the stability of adhesive joints substrate. (E) Variation in adhesive energy generated during pull-off of (Abdel Wahab, 2012; Brewis et al., 1990; Petrie, 2007; Tan et al., synthetic microthread with different capillary numbers. Adapted and reprinted 2008; White et al., 2005). Most of the synthetic adhesives fail when a with permission from Sahni, V., Labhasetwar, D. V. and Dhinojwala, A. (2012). Spider silk inspired functional microthreads. Langmuir 28, 2206-2210. crucial RH is exceeded (Petrie, 2007; Tan et al., 2008). Therefore, it Copyright 2012 American Chemical Society. This shows that it is possible to would be desirable to have synthetic adhesives that can either resist fabricate microthreads that in many ways mimic the appearance and changes in RH and continue to strongly bind surfaces or respond with performance of spider viscous threads. Journal of Experimental Biology

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Conclusions and outlook Competing interests Viscous thread adhesion relies heavily on water for both effective The authors declare no competing or financial interests. spreading of the adhesive glycoproteins and elasticity of the underlying axial thread. Water content also influences the Plateau– Funding National Science Foundation grant IOS-1257719 supported our research on viscous Rayleigh instability that determines the final size and spacing of glue thread hygroscopicity and the preparation of this Review. droplets. These features act synergistically to generate substantial adhesion as viscous threads deform in a suspension bridge-like References pattern while detaching from a variety of surfaces. Some of this water Abdel Wahab, M. M. (2012). Fatigue in adhesively bonded joints: a review. ISRN can be obtained directly from the atmosphere when threads are first Mater. Sci. 2012, 1-25. Agnarsson, I., Boutry, C., Wong, S.-C., Baji, A., Sensenig, A. and Blackledge, spun, potentially resulting in a net gain of water by a spider when an T. A. (2009). Supercontraction forces in spider dragline silk depend on rate of orb web is taken down and its silk ingested. Most orb webs are spun humidity change. Zoology 112, 325-331. under humid conditions, in the late evening or early morning, so that Amarpuri, G., Chaurasia, V., Jain, D., Blackledge, T. A. and Dhinojwala, A. (2015a). Ubiquitous distribution of salts and proteins in spider glue enhances minimal hygroscopicity is likely to be necessary for droplet spider silk adhesion. Sci. Rep. 5, 9053. formation and adhesion (Blackledge et al., 2009a). However, we Amarpuri, G., Zhang, C., Diaz, C., Opell, B. D., Blackledge, T. A. and Dhinojwala, hypothesize that increased thread hygroscopicity was necessary to A. (2015b). Spiders tune glue viscosity to maximize adhesion. ASC Nano 9, optimize thread adhesion as orb weavers diversified to occupy 11472-11478. Anderson, C. M. and Tillinghast, E. K. (1980). GABA and taurine derivatives on the habitats where humidity drops during the course of a day. Thus, adhesive spiral of the orb web of Argiope spiders, and their possible behavioural natural selection tuned the composition of LMMCs in a droplet’s significance. Physiol. Entomol. 5, 101-106. outer aqueous layer to meet this challenge (Townley and Tillinghast, Ayoub, N. A., Garb, J. E., Tinghitella, R. M., Collin, M. A. and Hayashi, C. Y. 2013) and to maintain glycoprotein structure and enhance its surface (2007). Blueprint for a high-performance biomaterial: full-length spider dragline silk genes. PLoS ONE 2, e514. interactions (Liao et al., 2015). However, this is largely based on Bai, H., Ju, J., Zheng, Y. and Jiang, L. (2012). Functional fibers with unique investigation of a few temperate species of spiders and three key wettability inspired by spider silks. Adv. Mater. 24, 2786-2791. questions remain about viscid thread hygroscopicity. First, what Blackledge, T. A. (2011). Prey capture in orb weaving spiders: are we using the best metric? J. Arachnol. 39, 205-210. about species in consistently arid or humid habitats such as deserts Blackledge, T. A. and Hayashi, C. Y. (2006). Unraveling the mechanical properties and rainforests? Do their glues perform similarly or show distinct of composite silk threads spun by cribellate orbweaving spiders. J. Exp. Biol. 209, LMMCs compositions? Second, can individual spiders control 3131-3140. LMMCs composition physiologically to tailor thread structure and Blackledge, T. A. and Zevenbergen, J. M. (2006). Mesh width influences prey retention in spider orb webs. Ethology 112, 1194-1201. adhesion under different physiological conditions? Finally, did the Blackledge, T. A., Scharff, N., Coddington, J. A., Szüts, T., Wenzel, J. W., hygroscopicity system arise to help spiders conserve water Hayashi, C. Y. and Agnarsson, I. (2009a). Reconstructing web evolution and resources after viscid glue was already being produced (e.g. the spider diversification in the molecular era. Proc. Natl. Acad. Sci. USA 106, ancestral condition was for orb spiders to exude wet sticky secretions 5229-5234. Blackledge, T. A., Scharff, N., Coddington, J., Szüts, T., Wenzel, J. W., Hayashi, from their aggregate glands) or as a mechanism to improve adhesion C. Y. and Agnarsson, I. (2009b). Spider web evolution and diversification in the (Opell et al., 2011b; Piorkowski and Blackledge, 2017) with spiders molecular era. Proc. Natl Acad. Sci. USA 106, 5229-5234. adding LMMCs to dry adhesive secretions for some other functional Blamires, S. J. (2010). Plasticity in extended phenotypes: orb web architectural responses to variations in prey parameters. J. Exp. Biol. 213, 3207-3212. benefit? Blamires, S. J., Sahni, V., Dhinojwala, A., Blackledge, T. A. and Tso, I. M. (2014). Our current model of the evolution of viscous thread Nutrient deprivation induces property variations in spider gluey silk. PLoS ONE 9, environmental responsiveness relies entirely on describing e88487. variation in LMMCs composition. The amino acid sequence of Blamires, S. J., Tseng, Y. H., Wu, C. L., Toft, S., Raubenheimer, D. and Tso, I. M. (2016). Spider web and silk performance landscapes across nutrient space. Sci. only one glycoprotein has been characterized and details of this Rep. 6, 26383. molecule’s three-dimensional structure and adhesion are not well Blamires, S. J., Hasemore, M., Martens, P. J. and Kasumovic, M. M. (2017). Diet- understood. Thus, the model we present here is clearly an induced co-variation between architectural and physicochemical plasticity in an extended phenotype. J. Exp. Biol. 220, 876-884. oversimplified view. For instance, how much of the variation in Bond, J. E. and Opell, B. D. (1998). Testing adaptive radiation and key innovation the environmental responsiveness of different species’ glue is hypotheses in spiders. Evolution 52, 403-414. explained by interactions between LMMCs and variation in Bott, R. A., Baumgartner, W., Bräunig, P., Menzel, F. and Joel, A. (2017). glycoprotein sequence? Future investigation should also focus on Adhesion enhancement of cribellate capture threads by epicuticular waxes of the insect prey sheds new light on spider web evolution. Proc. R. Soc. B 284, understanding how LMMCs directly interact the glycoproteins to 20170363. plasticize them and how this influences adhesion. Indeed, selection Boutry, C. and Blackledge, T. A. (2013). Wet webs work better: humidity, for optimal glycoprotein secondary structure may be as important as supercontraction and the performance of spider orb webs. J. Exp. Biol. 216, selection for optimal aqueous layer hygroscopicity. 3606-3610. Brewis, D. M., Comyn, J., Raval, A. K. and Kinloch, A. J. (1990). The effect of The use of LMMCs to recruit water and control the self- humidity on the durability of aluminium–epoxide joints. Int. J. Adhesion Adhes. 10, organization of a hierarchically structured adhesive thread is simple 247-253. in concept and therefore translatable to synthetic models. However, Chen, Y. and Zheng, Y. (2014). Bioinspired micro-/nanostructure fibers with a water collecting property. Nanoscale 6, 7703-7714. we still do not understand the specific functions of individual Choresh, O., Bayarmagnai, B. and Lewis, R. V. (2009). Spider web glue: two LMMCs and the mechanisms by which they plasticize the adhesive proteins expressed from opposite strands of the same DNA sequence. glycoproteins. In addition to optimizing the performance of Biomacromolecules 10, 2852-2856. synthetic adhesives, such research will also provide a powerful Coddington, J. A. (1989). Spinneret silk spigot morphology: evidence for the monophyly of orb-weaving spiders, Cyrtophorinae (Araneidae), and the group tool to test hypotheses about specific aspects of viscous thread Theridiidae plus Nesticidae. J. Arachnol. 17, 71-96. function and spider web evolution. Collin, M. A., Clarke, T. H., Ayoub, N. A. and Hayashi, C. Y. (2016). Evidence from multiple species that spider silk glue component ASG2 is a spidroin. Sci. Rep. 6, 21589. Acknowledgements Crews, S. C. and Opell, B. D. (2006). The features of capture threads and orb-webs We are grateful to two reviewers whose comments and suggestions allowed us to produced by unfed Cyclosa turbinata (Araneae: Araneidae). J. Arachnol. 34, improve the clarity and completeness of this Review. 427-434. Journal of Experimental Biology

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