Silks and silk-producing organs of Neotropical tarantula Avicularia metallica ( Araneae , Mygalomorphae , Theraphosidae )

Silks and silk-producing organs of the theraphosid species Avicularia metallica were studied using scanning electron microscopy. The spinning apparatus is made up of two pairs of spinnerets located at the end of the ventral side of the opisthosoma. Both pairs of spinnerets are equipped with spigots (modified setae), i.e. external outlets of silk-producing glands which, in the case of posterior lateral spinnerets, are present on all three segments. The secreted silk, which hardens when exposed to air, is processed by movements of spinnerets and the opisthosoma. An investigation of spinning activity revealed two different manners in which silk is affixed to the ground: (1) by smearing silk secretion directly onto the surface of the substratum; or (2) by attaching silken fibers onto a layer of adhesive silk of attachment fields. The fibers connecting the walls of tubular shelters to the silk of attachment fields are essentially bundles of parallel nanofibrils. The connection between multifibrillar connecting fibers and the adhesive silk of attachment fields is, in essence, “silk-to-silk” connection. Nanoglobules (spherical subunits) are the basic microstructural blocks in the studied silk materials irrespective of whether the fibrils are a part of the tube, connecting fibers, or attachment fields. SEM images showed that the liquid silk, running through spigot ducts, has two components, which do not mix as they leave the spigots. The peripheral component of the solidified protein mixture surrounds the central component, which has a granular appearance.


Introduction
Evolution of the order Araneae most likely occurred along three major evolutionary lines, currently represented by the infraorders Mesothelae, Mygalomorphae, and Araneomorphae (Coddington & Levi 1991).The infraorders exhibit differences in the structure of some important organs, the most remarkable of which is the silk-producing organ.The silk-spinning apparatus of spiders consists of spinnerets and silk spinning glands located in the opisthosoma.These glands originate from ectodermal invaginations on the embryonic spinneret limb buds, in relation to the morphogenesis of these buds (Hilbrant & Damen 2015).The saclike silk glands of a spider are lined by a secretory epithelium that is supported by a basal lamina and that secretes silk secretion into the glandular lumen (Kovoor 1987).Silk spinning glands supply sets of spigots (nozzles), which are generally regarded as modified setae (Bond 1994).The epithelial cells are capable of producing different types of silk secretions which consist of liquid fibroin proteins.Spider silks are viscoelestic polymers that change their material properties as they are stretched (Blackledge 2012).The silk fibers that are spun from these secretions are used in different and unique ways such as draglines, snares,

Research Article
egg sacs and shelters (Foelix 2011).Although the molecular structure of silk fibers is fairly well understood, the hierarchical organization and complexity of constituents in the silk fibers remain poorly understood (Cranford 2013;Sampath & Yarger 2015).
Spinnerets are highly modified opisthosomal appendages.Kautzch (1910) found that in the spider Agelena labyrinthica (Clerck, 1757) respiratory organs have developed extremities on the bases of the 8 th and 9 th body segments, and external spinning organs on the 10 th and 11 th segments.Yoshikura (1955) confirmed Kautzch"s conclusion in his study of the spider Heptathela kimurai Kishida, 1920 (Mesothelae).In the paper above, Yoshikura also found that the two lateral pairs of spinnerets appear first, followed by the bases of the 10 th and 11 th segments of extremities; he further found that the two median pairs become separated only in the postembryonic stage.The oldest known silk-producing spigots date back to the Middle Devonian of Gilboa, New York, USA (Shear et al. 1989).The spinneret found was very much like the posterior median spinnerets of the spider infraorder Mesothelae (Selden et al. 2008).
The Mesothelae are the most archetypal of recent spiders known (Haupt 2003).The most thoroughly described genera are Liphistius Schiödte, 1849, from South and South-East Asia, and Heptathela Kishida, 1923, which includes 33 species from Japan, Vietnam and China (World Spider Catalog 2016).Liphistius has four pairs of spinnerets: two pairs of multisegmental lateral and two pairs of monosegmental median.In Heptathela kimurai the posterior and the median pairs of spinnerets merge to form a single unpaired spinneret without spinning capabilities (Haupt & Kovoor 1993;Haupt 2003).Except for the Mesothelae, four pairs of spinnerets are never present, since an anterior median pair has not developed.
The Araneomorphae, known as "true spiders", with the largest number of species, possess three pairs of spinnerets, namely the anterior lateral (ALS), posterior median (PMS), and posterior lateral spinnerets (PLS).In some spiders of the infraorder Araneomorphae, the spinning apparatus includes a functional homolog of anterior median spinnerets called the cribellum.In other true spiders, the alternative homolog of anterior lateral spinnerets is nonfunctional colulus (Kovoor 1987).
Most reviews, books and journal articles on spider silk production, its molecular composition or physical/mechanical processing, have focused on understanding the dragline silk of araneomorphs.Dragline silk is an extracellular fibrous protein called spidroin, which exhibits a unique combination of strength and toughness (Tian et al. 2013).Taut dragline fibers are connected to the substratum by means of attachment discs.Anchored to the substratum by attachment discs, the "silk track" allows the spider to return safely to the starting point after a thrust at prey or following a free fall; it also allows spiderlings to maintain contact with the parental web.The same silk is used as a bridge lines and ballooning threads (Blackledge 2012).Dragline silk is produced by the secretory activity of a pair of major ampullate glands (Glandulae ampullaceae major), while the material of attachment discs is secreted by piriform glands (Glandulae piriformes) (Kovoor, 1987).The spigots of both types of glands are located on the surface of anterior lateral spinnerets.
In the majority of recent spiders of the infraorders Mygalomorphae and Araneomorphae, the spinnerets are located at the posterior end of the abdomen.One of the prominent features of spiders of the infraorder Mygalomorphae, which, like Mesothelae, often live in tubular silken shelters, is the tendency to a reduction in the number of spinnerets, which undoubtedly results in a limited diversity of spinning glands, spigots, and produced silk.In all mygalomorphs the anterior median spinnerets are completely extinct, and a great number of these spiders also have reduced or nonfunctional anterior lateral spinnerets.
In total 46,058 spider species belonging to 3,988 genera and 114 families are known (World Spider Catalog, 2016).Of this number, 2,720 species belong to the infraorder Mygalomorphae.The ultrastructure of silk produced by the family Theraphosidae (common name: tarantulas, or baboon spiders), which includes the largest recent spiders and which is the most numerous family of the infraorder Mygalomorphae with its Ecol. Mont., 7, 2016, 313-327 315 132 genera and 953 species (World Spider Catalog, 2016), has not yet been investigated.The spinning apparatus of Theraphosidae consists of two pairs of spinnerets in which aciniform spinning glands supply one type of spigots (Murphy & Roberts 2015: 57, 220-221).The family is divided into ten subfamilies, and the genus Avicularia Lamarck, 1818, belongs to the subfamily Aviculariinae, which is endemic to South America.Like most spiders of the genus Avicularia, the species under investigation, Avicularia metallica, Ausserer, 1875, is one of the arboreal and rain forest species found in silken tube retreats in crevices of trees, in holes under bark or under epiphytes and on rain forest vegetation (Jocqué & Dippenaar-Schoeman 2006).This species, called metallic "whitetoe", or metallic "pinktoe" (with a diagonal leg span of approximately 13-15 centimetres), is distributed mainly in Columbia and Suriname (World Spider Catalog, 2016).
The aim of the research, the results of which are presented in this paper was (1) to study the morphology of the spinnerets, in particular their spigots through which silk is emitted on their surface and (2) to use scanning electron microscopy (SEM) to describe the ultrastructure of the silk fibers used by the theraphosid species Avicularia metallica to build its tube-like shelters.The article also describes the spinning behavior and the movements of the spinnerets made when the silk fibers are secreted.Data presented in this paper may contribute to a better understanding of the hierarchical organization of constituents in the silk fibers of the Mygalomorphae.

Materials and keeping spiders in laboratory conditions
All specimens of Avicularia metallica studied (Fig. 1) were obtained from tarantula breeders in the Czech Republic.The silk-producing organs and silk micro-and ultrastructure were observed between December 2011 and January 2015.The group of specimens under investigation comprised five adult females, three males and five 5 th instar nymphs.The body length (prosoma + opisthosoma) of the adult spiders ranged from 58 to 62 mm, while the nymphs were up to 18 mm long.
Adult specimens were kept in glass insectaria measuring 28 × 25 × 30 cm.The floor of the insectariums was covered with a layer of peat moss approx.5 cm thick and a few pieces of oak bark.The nymphs were kept under the same conditions in smaller glass containers measuring 10 × 8 × 12 cm.The spiders and their spinning behavior were observed with a Dino-Lite Digital Microscope.For the observation of living spiders it was necessary to use KL 1500 LCD ZEISS cold light sources.
Voucher specimens have been deposited at the Department of Biology, Faculty of Science, J.E. Purkinje University in Ústí nad Labem, Czech Republic.

Method of obtaining samples of silk for scanning electron microscopy
For the mechanical harvesting of intact silk samples suitable for investigation of their micro-, ultra-and nanostructure, each spider was kept for a certain time inside thin-walled transparent plastic containers measuring 15 × 20 × 25 cm.After the spiders built a complete web, the plastic walls of the container were cut into small pieces (approx. 2 × 2 cm), and the silk attached to them was studied using SEM.

Scanning electron microscopy imaging equipment
For the imaging of spinnerets, spigots and the ultrastructure of silk fibers, the following equipment was used: a TESCAN SEM Microscope (Fig. 4A-D), XL 30 ESEM Environmental Scanning Electron Microscope (Fig. 7A-C) and UHR-SEM Zeiss Ultra Plus Microscope (Figs. 5A-C, 6A-D, 8A-D, 9A-B), operated at low acceleration voltage of 1 kV.For SEM studies, the spiders were killed by chloroform, dehydrated in ethanol, and processed via critical point drying using the carbon dioxide.Prior to observation, samples were coated with a 1 nm thick layer of platinum.A Quorum Q R150ES sputter coater was used for the coating.Measurement/image analysis was carried out using SmartSEM software.Shelters built by all the specimens kept in laboratory conditions had the appearance of tubes (Figs.2A-B, 3), measuring, for adults, up to 30 cm in length and 45-50 mm across.Between the walls of the tube and the wall of the insectarium (or objects near the tube) have always been stretched bundles of contacting fibers (Fig. 3).These fibers not only maintain the shape of the tube but also keep it passable at all times.The fibers connecting the wall of the tube to the surrounding objects or to the wall of the container were in all cases attached by means of adhesive silk of attachment fields (Figs.2B, 3).The spiders not only take refuge in the tube, but they also molt in it.This type of web does not serve as a snare.

Spinnerets and their role in the processing of emitted silk
The spinning apparatus in adults and juveniles is made up of two pairs of spinnerets located at the end of the ventral side of the opisthosoma (Figs.1B, 4A-B).Not only anterior median, but also anterior lateral spinnerets are completely reduced; only the monosegmental posterior median (PMS) and three-segmented posterior lateral spinnerets (PLS) are functional.Both pairs of spinnerets are equipped with spigots (Fig. 4C-D), i.e. external outlets of silk-producing glands which, in the case of PLSs, are present on all three segments.All segments of spinnerets were densely covered with hairs, which often obscured the spigots, making observation difficult.
The observed tarantulas do not use their legs to process the silk during shelter construction.The secreted silk, which hardens in the air, is processed by movements of the spinnerets and the opisthosoma.As the opisthosoma moves over the substratum, the emitted silk connects different points on the surface of objects in the space where the shelter is being built.Posterior median and posterior lateral spinnerets rarely touch each other during spinning activity.Most of the time, the movements of spinnerets of the same pair are asynchronous and their segments carrying spigots point in different directions while they emit silk.In such cases the spiders touch the substratum with both spinnerets, or with only one of them.The number of spinnerets/spigots emitting silk at any one time affects the number of nanofibrils in fibers and, subsequently, their thickness.An investigation of the spinning activity of Avicularia metallica revealed two different manners in which silk was affixed to the surface of the glass or plastic walls of the breeding container: (1) by smearing silk secretion directly onto the surface of the substratum; or (2) by attaching silken fibers onto the adhesive silk layer of attachment fields.

Ultrastructure of spigots and their role in silk production
Each spigot consists of two parts, basal (i.e.socket) and terminal (i.e.shaft) (Fig. 5A-C).In comparison with the setae, which surround them on the surface of spinnerets, the sockets are high, flattened along their sides, with clearly distinguishable contours of the ductus which lead into them, feeding liquid silk into the shafts.The shafts are long, slender and slightly bent distally.They have an opening at the end (Fig. 5B), which emits secretion, initially liquid, but fast hardening when exposed to air.SEM images showed that the liquid silk, running through spigot ducts, has two components, which do not mix as they leave the spigots (Fig. 5C).The peripheral (S1) component of the solidified protein mixture surrounds the central (S2) component, which has a granular appearance unlike S1.When the spinnerets rub against the ground and when the terminal parts of spigots come into contact with the substratum, each spigot extrudes a separate fibril (= nanofibril), i.e. a separate "silk trail" (see below -Silks of attachment fields).It is only after the spinnerets are lifted and put into motion above the substratum that the nanofibrils are joined laterally.This produces bundles of parallel nanofibrils, which form the basis of thick connecting fibers.

Silks of shelters
The densely spun walls of the adults" tube-like shelters were largely made up of nanofibrils approx.140-220 nm in diameter (Fig. 6A).In certain places, especially on the surface of the tube near its entry opening, the nanofibrils were thicker (500-600 nm) (Figs.6B-C).In addition to nanofibrils, which are used to construct the wall of the tube, bundles of parallel nanofibrils of roughly the same thickness (130-140 nm in diameter) can be seen on its surface.These bundles are the basic material of the connecting fibers (Fig. 6D), which connect the surface of the tube wall and the silk of attachment fields.The longest and thickest connecting fibers are found around the entry opening of the shelter (see Fig. 3).Nanoglobules were the basic microstructural elements (Fig. 6A) in the silk materials studied, irrespective of whether the fibrils are a part of the tube, connecting fibers, or the attachment fields.

Silks of attachment fields
The silk of attachment fields (Fig. 2A-B) provides an adhesive underlying layer, onto which the spider attaches connecting fibers (i.e.bundles of nanofibrils), stretched between the tube and the substratum (Fig. 3).In laboratory conditions, the spiders used this silk to cover the smooth glass or plastic walls of breeding containers in places where they subsequently built shelters.Liquid silk is released from spigots after the distal ends of their shafts touch the substratum or objects, as they move on their surface; the liquid silk hardens as the spinnerets move either on the surface of the substratum or above it.In the former case, spigots leave silk "trails" on the substratum in the form of separate nanofibrils which have hardened and remained glued onto the surface of the substratum along their entire length.In this way, parts of objects are covered  In some places, bundles of nanofibrils are visible, having approximately the same thickness; when observed with low magnification or with the naked eye, they have the appearance of the thick connecting fibers connecting the surface of the tube wall with the adhesive silk of attachment fields (see Fig. 3).D -A connecting fiber connecting an attachment field with tube of shelter.BFbundle of nanofibrils, CFconnecting fiber.
with an adhesive coating, onto which the spiders attach the fibers of their shelters (fig.7A-C).This type of silk coating is the material substance for "attachment fields", although it can also be found outside shelters on vertical glass walls of the insectarium in spots where the spiders wait for prey or rest.The connection between connecting fibers and the adhesive silk (Fig. 7A) of attachment fields is a "silk-to-silk" connection.Prior to contact with the adhesive silk, the thick bundles of nanofibrils (Fig. 7B-C) leading from the tube to the ground ramify progressively into branches made up of a decreasing number of nanofibrils, which become attached to the adhesive surface of the ground either as thin fibers or separate nanofibrils.

Nongranular and granular secretion and formation of the ultrastructure of fibers in the process of liquid silk solidification
As explained above, silk leaving the spigots has two components which do not mix as they pass through the spigots (see Fig. 5C).An SEM study of samples of silk produced by spiders on the surface of plastic breeding containers proved the presence of spots of amorphous, nongranular secretion with fibrils of diverse lengths leading from them as their continuation (Fig. 8A-D).These fibrous projections were made up of fibroin material, concentrated in small patches of granular secretion (Fig. 9C) and possessing a granular (Fig. Figure 7A-C.SEM micrographs of attachment field silk: A -Cut-out section of plastic container, covered with silk of attachment fields -AFS.Places with the highest concentration of nanofibrils appear in the image as whitish spots without clear borders.This silk constitutes an adhesive foundation layer onto which the spider attaches connecting fibers, Battachment of a connecting fiber (CF) to the substratum covered with adhesive silk.Before joining with the silk of attachment fields, the thick connecting fibers ramify into several thinner fibers with fewer nanofibrils.As the fibers progressively ramify before they are anchored in the AFS-covered substratum, the bundles become gradually less compact until separate nanofibrils remain.Cenlarged view of framed section in B. Ecol. Mont., 7, 2016, 313-327 323 8D), or nanoglobular, ultrastructure.Such spots of amorphous secretion connected with fibers of nanoglobular structure (Fig. 9A-B) can be found on the surface of breeding containers in large numbers.
They occur where the spiders tend to stay in one place without building three-dimensional webs there.

Discussion
Like the other Theraphosidae, Avicularia metallica has only two pairs of spinnerets: monosegmental posterior median and three-segmented posterior lateral spinnerets.Two pairs of spinnerets are a characteristic feature of all theraphosids studied to date, although spiders of the infraorder Mygalomorphae include families which have, like the Araneomorphae, developed three pairs of spinnerets (for example, Atypidae, Hexathelidae, Mecicobothriidae).On the other hand, a single functional pair of posterior lateral spinnerets is characteristic of spiders of the family Nemesiidae, whose small, monosegmental posterior median spinnerets have no spigots at all (Glatz 1972).
A comparison of the spinning activity and web weaving methods of spiders of all three infraorders leads to the conclusion that evolution of webs, their diversity and their ability to fill various habitats has been affected most of all by the presence or absence of anterior lateral spinnerets and related glands, i.e. major ampullate and piriform silk glands.This fact should be taken into consideration when formulating hypotheses about the original function of spider silk and web evolution.The reduction of anterior lateral spinnerets, which led to the loss of the ability to produce draglines and attach them to the ground by means of attachment discs is probably the most important evolutionary change that has affected the spinning activity of spiders of the infraorder Mygalomorphae.As a result of this loss, the Theraphosidae and most likely other Mygalomorphae, too, have developed another method of affixing their silk threads to the ground.In the case of Avicularia metallica, the silken fibers of the three-dimensional tube-like shelters were connected to the surface of surrounding objects by means of the adhesive silk of attachment fields.The adhesive silk was applied onto the surface of objects by rubbing the spinnerets against the substratum, while the spigots directly touched the substratum, leaving silk "trails" in the form of nanofibrils glued to the substratum.
Very little is known about tarantulas" silk production and silk ultrastructure.Therefore, it is impossible to establish to what extent the ultrastructure of silks and the connection of fibers to the surface of objects observed in the theraphosid Avicularia metallica matches or differs from those observed in other families with different numbers of spinnerets and different degrees of spinning apparatus reduction.The role of anterior lateral spinnerets of the family Hexathelidae has not been investigated as yet, and it would certainly be important to explore the function of these spinnerets and the silk they emit, because, unlike most mygalomorphs, they are equipped with spigots not only as spiderlings but also in adult stages (Glatz 1972).Glatz has found that adults" bi-segmented ALSs are equipped with 6-8 spigots that are concentrated, as in the case of spiders of the infraorder Araneomorphae, on a spinning field of the apical segment.These spigots are connected to small glands which are similar to the piriform glands of the Araneomorphae, as, like those glands, they consist of two parts and their epithelia produce both a basophilic and an acidophilic secretion (Glatz 1972(Glatz , 1973)).However, the presence of ampullate glands and their spigots has not been established in Hexathele hochstetteri or in other Mygalomorphae.The distribution of spigots on the surface of PMSs and PLSs is similar to that in Theraphosidae, i.e. they are dispersed between hairs on all segments.
Silk similar to adhesive silk was also found on the vertical walls of breeding containers in places where specimens of A. metallica exposed themselves to sunrays, waited for prey or rested.Such places turned out to be a great interest in terms of the further study of the role of nongranular and granular secretions in the formation of the ultrastructure of fibers, supramolecular interactions and solidification of silk left there by sitting spiders.
An important feature of the spinning activity of Avicularia metallica (and probably all theraphosids) is the asynchronous movement of posterior median and posterior lateral spinnerets belonging to the same pair as well as the processing of the emitted silk only by movements of the opisthosoma and spinnerets.The posterior median and, above all, the long posterior lateral spinnerets of Avicularia can move asynchronously in all directions, each touching the substratum independently.A similar method of handling the hardening silk extruded from the spigots of posterior median and posterior lateral spinnerets is employed by haplogyne spiders of the family Dysderidae, belonging to the infraorder Araneomorphae (Hajer et al. 2013).The effective working of the spinnerets is, as in other spiders, enhanced to a great extent by the movements of the opisthosoma itself (Foelix 2011) and also by the considerable mobility of posterior lateral spinnerets, whose terminal segments are usually digitiform in mygalomorph spiders.So far, research into the spinning activity of spiders has brought little information on the behavioral capabilities of spider spinnerets or the functional significance of spigot placement on the spinnerets for processing different types of silk (Eberhard 2010).Like Avicularia metallica, mygalomorph spiders Antrodiaetus unicolor (Antrodiaetidae) are also equipped with two pairs of spinnerets.Adults of both species have only one type of silk glands and spigots, which are grouped into four clusters, each cluster serving one of the four spinnerets (Palmer et al. 1982).The cited paper describes their spigots as long, slender, and slightly bent distally.Although all gland cells are structurally similar, each gland simultaneously produces (as in A. metallica) two different secretory products: the secretion of the distal region being rich in basic protein and sulfhydryl groups, and the proximal region secretion being an acidic protein containing a high concentration of histochemically demonstrable C-terminal carboxyl groups (Palmer et al. 1982).
The silk-producing organs of several specimens representing at least three species of the mygalomorph genus Euagrus were examined by Palmer (1985) by means of a variety of absorption and fluorescent histochemical techniques.The silk glands were arranged in four groups, each serving one of four spinnerets.As in the case of Avicularia metallica, the spigot morphology of Euagrus was uniform, consisting of a long, slender shaft of slightly variable length emerging from an enlarged sac-shaped base.
SEM study of the silk of Avicularia metallica confirmed that nanoglobules are the basic microstructural blocks in the theraphosid silk studied.In terms of thickness, the fibrils used by Avicularia metallica to construct the silken tube are heterogeneous, but in terms of ultrastructure, they are homogeneous.Yet the thickness of nanofibrils whose bundles formed the connecting fibers connecting the tube with the surface of surrounding objects was always the same.It may be assumed that research into spider silk in the near future will retain its focus on the silk of the araneomorph orb-weaver family Araneidae.To many observers the symmetrical orb webs spun by many members of this group epitomize engineering skill and natural beauty (Griswold et al. 1998).
However, spider silk is also antimicrobial (Gomez et al. 2011), hypoallergenic and completely biodegradable (Römer & Schiebel 2008), which makes the case for research of silk spiders with a low diversity of spinning glands, i.e. those which have been neglected to date and the research of whose spinning apparatus, silk production and spinning behavior may help to resolve their evolution.The silk of mygalomorph spiders remains to be investigated.

Figure
Figure 1A-B.Adult female Avicularia metallica: Aa female sitting on a leaf of Dwarf Cavendish banana Musa acuminata.Bthe same female kept inside a glass insectarium.The female leans against the glass wall both with its feet and spinnerets at the end of its opisthosoma.SPspinnerets, I. -IV.first to fourth pairs of legs.

Figure
Figure 2A-B.Macrostructure of shelter and attachment fields: Amid-section of silken tube.Arrows indicate where the tube is attached to the substratum by means of attachment fields (AF).Bdetailed view of an attachment field.

Figure
Figure 4A-D.SEM micrographs of spinnerets: Aoverview of ventral side of a nymph.Benlarged view of framed section in A: posterior part of ventral side of opisthosoma with two pairs of spinnerets.Csurface of posterior lateral spinnerets with spigots, partly covered by adjacent hairs (setae).Location of spigots is marked with arrows.Ddetailed view of surface of spinnerets with spigots, hairs and silk fibrils.CHchelicerae, HAhairs , PEpedipalps, PMSposterior median spinnerets, PLSposterior lateral spinnerets, SIsilk, SPspinnerets, SPIspigots, I. -IV.first to fourth pairs of legs.

Figure
Figure 5A-C.Spinneret silk spigot ultrastructure: Atwo spigots of posterior lateral spinnerets.Benlarged view of framed section in A: opening at the end of shaft, from which silk secretion is extruded, Copening at the end of shaft, filled with solidified silk secretion.The secretion has two components.Peripheral (S1) component of solidified protein mixture surrounds the central (S2) component, which unlike component S1, has a granular appearance.SPIspigots, basbasal part (socket), termterminal part of spigot (shaft).

Figure
Figure 6A-D.-SEM micrographs of shelter silk: A -Detailed view of silk nanofibrils of the same ultrastructure, approx.140-220 nm in diameter, which constitute wall of the tube.Nanoglobules are the basic ultrastructural elements of the depicted silk.B -Detailed view of nanofibrils of various thicknesses, scanned and measured elsewhere in the wall of the tube.C -Nanofibrils and connecting fibers on surface of the tube.In some places, bundles of nanofibrils are visible, having approximately the same thickness; when observed with low magnification or with the naked eye, they have the appearance of the thick connecting fibers connecting the surface of the tube wall with the adhesive silk of attachment fields (see Fig.3).D -A connecting fiber connecting an attachment field with tube of shelter.BFbundle of nanofibrils, CFconnecting fiber.

Figure
Figure 8A-D.Amorphous and fibrous structures in attachment fields: A -Spot of amorphous (i.e.nongranular) secretion on the surface of plastic wall with fibrils of various lengths leading from it in numerous places.Benlarged view of framed section in A. C -Detailed view of surface of the spot.Granular secretion in center of image is the main structural material of a fibril.Ddetailed view of fibril (150-170 nm in diameter) with characteristic globular ultrastructure, formed on the underlying layer of nongranular component of silken spot.GSgranular secretion, NGSnongranular secretion, NFnanofibril.

Figure
Figure 9A-B.Connection between nongranular silken secretion and fibrils: A -Solidified drop of amorphous secretion joined with fibrils.Benlarged view of ultrastructure of silk where the two types of secretion connect.