© 2017. Published by The Company of Biologists Ltd | Journal of Experimental Biology (2017) 220, 2243-2249 doi:10.1242/jeb.157594 RESEARCH ARTICLE Nanofibre production in spiders without electric charge Anna-Christin Joel1,* and Werner Baumgartner2 ABSTRACT attraction of prey but for the formation of its own structure, using the Technical nanofibre production is linked to high voltage, because repulsion between two identically charged objects (Opell, 1995a; nanofibres are typically produced by electrospinning. In contrast, Joel et al., 2015; Kronenberger and Vollrath, 2015). Prey is captured ’ spiders have evolved a way to produce nanofibres without high here by a combination of hygroscopic forces, van der Waals forces voltage. These spiders are called cribellate spiders and produce and an entanglement of the prey in a wool-like mat of nanofibres nanofibres within their capture thread production. It is suggested that (cribellate fibres) surrounding two larger stabilizing axial fibres their nanofibres become frictionally charged when brushed over a (Opell, 1994; Hawthorn and Opell, 2003). continuous area on the calamistrum, a comb-like structure at the The cribellate fibres shape the outer structure of the capture thread metatarsus of the fourth leg. Although there are indications that with two characteristic regions: the puff with a larger diameter than electrostatic charges are involved in the formation of the thread the intermediate zones, clearly separating two puffs with a structure, final proof is missing. We proposed three requirements to constriction (Fig. 1) (Peters, 1984; Opell, 1989; Joel et al., 2015). validate this hypothesis: (1) the removal of any charge during or after It is commonly assumed that the adhesive cribellate fibres are kept thread production has an influence on the structure of the thread; (2) separate within the puff by being charged uniformly during their the characteristic structure of the thread can be regenerated by extraction, leading to a repulsion of the fibres (Peters, 1984; Sahni charging; and (3) the thread is attracted to or repelled from differently et al., 2011; Kronenberger and Vollrath, 2015). During charged objects. None of these three requirements were proven true. the production of the thread, the nanofibres are extracted from the Furthermore, mathematical calculations reveal that even at low cribellum, a spinning plate with up to 40,000 spigots anterior to charges, the calculated structural assembly of the thread does not the spinnerets (Bertkau, 1882; Foelix and Jung, 1978). To assemble match the observed reality. Electrostatic forces are therefore not all these fibres to one functional thread, the spiders deploy a highly involved in the production of cribellate capture threads. sophisticated movement of the spinnerets as well as the fourth pair of legs, finally wrapping the sheet of nanofibres around the axial KEY WORDS: Cribellar, Processing, Uloboridae, Filistatidae, Silk, fibres (Joel et al., 2015, 2016). On the metatarsus of the fourth leg, Protein cribellate spiders bear a comb-like row of specialized setae, the calamistrum. It is assumed that the charge is transferred to the INTRODUCTION cribellate fibres by triboelectrification when they are brushed over a We all have experienced the effects of rubbing a plastic balloon continuous area composed of the specialized setae of the against our hair: the single hairs are repelled from each other, but calamistrum (Fig. 1B) (Peters, 1984; Kronenberger and Vollrath, attracted towards the balloon. This is induced by Coulomb forces, 2015; Joel et al., 2016). also called electrostatic forces, which are caused by frictional This kind of nanofibre production involving only low electric charging in this example (triboelectrification). Even without charge has attracted the interest of researchers, as technical friction, electrons drift from one material to the other whenever nanofibres are typically produced by electrospinning, involving two surfaces are in contact, and after separation, the materials high tension (Teo and Ramakrishna, 2006; Kronenberger and become oppositely charged. Most materials are easily discharged Vollrath, 2015). From the biological perspective, evidence for a again by protons and electrons occurring in the air. The final charging of cribellate fibres by the action of the calamistrum is charging of an object hence depends not only on the physical and missing so far. However, there are indications that electrostatic chemical nature of the given material but also on the conductivity of charges are involved in the formation of the puffy structure: high the medium surrounding the object (Chang et al., 1995). humidity can increase the conductivity of air and hence remove Many biological systems use the attraction between oppositely charge. Indeed, the thread loses its characteristic structure after charged objects: this charging facilitates, for example, the landing of being exposed to the fine mist of a nebulizer (Zheng et al., 2010; pollen on the stigma during pollination by wind (Bowker and Elettro et al., 2015). Furthermore, spiders without calamistra Crenshaw, 2007) and also attracts the gluey capture threads of produce capture threads without puffs (Joel et al., 2015). This spiders to positively charged flying insects (Ortega-Jimenez and could indicate missing electrostatic charges to keep the fibres Dudley, 2013). Another type of capture thread, the cribellate capture separate, though the use of a nebulizer, i.e. fine droplets of water, thread, is even suggested to employ electrostatic forces not for the could also destroy the structure of the thread as a result of capillary forces or a reaction with the spider silk. The aim of this study was hence to resolve the involvement of Coulomb forces in the nanofibre 1RWTH Aachen University, Institute of Biology II, Worringerweg 3, 52074 Aachen, production of cribellate spiders. Germany. 2JKU Linz, Institute of Biomedical Mechatronics, Altenberger Straße 69, 4040 Linz, Austria. There are several ways to determine whether cribellate nanofibres are indeed electrostatically charged and whether such charging helps *Author for correspondence ( [email protected]) formation of a puffy structure: (1) one can try to regenerate the puffy A.-C.J., 0000-0002-7122-3047 structure of threads by charging them; (2) one can remove any charge by (i) making the surrounding conductive or (ii) making the fibres Received 2 February 2017; Accepted 3 April 2017 conductive; and (3) one can determine whether they are attracted to Journal of Experimental Biology 2243 RESEARCH ARTICLE Journal of Experimental Biology (2017) 220, 2243-2249 doi:10.1242/jeb.157594 A B Calamistrum Metatarsus 5 mm 200 µm C D E cf cf cf ax ax Puff iz 100 µm Puff iz 100 µm Puff iz 100 µm Fig. 1. The cribellate spider Uloborus plumipes. (A) An adult female in its resting position in the hub of its web. (B) Enlarged view of the calamistrum on the metatarsus of the fourth leg of the spider. Scanning electron microscope (SEM) image, sample coated with gold. (C–E) Capture threads of U. plumipes after different treatments (C, coated with gold; D, untreated; E, coated with carbon). SEM images. ax, axial fibre; cf, cribellate fibres; iz, intermediate zone. or repelled from differently charged objects. On the basis of these endangered or protected species and special permits were not three requirements, we aimed to determine whether electrostatic required. All applicable international, national and institutional forces are deployed during the formation of the cribellate thread. All guidelines for the care and use of animals were followed. experiments were performed with Uloborus plumipes (Uloboridae) whose capture thread formation has been best described to date Study animals (Fig. 1A). To evaluate the general validity of the findings, threads of Adult female Uloborus plumipes Lucas 1846 and Zosis geniculata another Uloboridae, Zosis geniculata, as well as the distantly related (Olivier 1789) were raised in larger terraria shared by several Amaurobiidae Amaurobius ferox, Desidae Badumna longinqua and spiders of the same species as a lab colony under room temperature the Filistatidae Kukulcania hibernalis were examined (Bond et al., (∼21°C), room humidity (∼30%) and northern European diurnal 2014; Fernández et al., 2014; Garrison et al., 2016). Capture threads rhythm. Each spider was able to build a web of its own. Once a of these species differ greatly in shape from those of Uloboridae week, spiders were fed with Drosophila melanogaster or juvenile (Fig. 2). Comparable results would indicate a conserved cribellate Acheta domestica. Water was provided once or twice per month by fibre production system in all cribellate spiders, whereas differences sprinkling the web. Threads of such sprinkled webs were not used would hint at a diverging mechanism involved in the formation of for further research. cribellate capture threads. Kukulcania hibernalis (Hentz 1842) were raised separately in 1 litre containers covered with gauze under elevated room MATERIALS AND METHODS temperature (∼26°C), normal room humidity and northern Ethics European diurnal rhythm. Once a week, spiders were fed with The species used in the experiments (Amaurobius ferox, Kukulcania juvenile A. domestica or bean weevils. Water was provided once or hibernalis, Uloborus plumipes and Zosis geniculata) are not twice per month by applying droplets near the burrow. Thread samples of Amaurobius ferox were collected near the Institute of High Frequency Technology