Chapter 3 Silk Glands of Araneid Spiders Selected Morphological and Physiological Aspects
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Edward K. Tillinghast and Mark A. Townley Department of Zoology, University of New Hampshire, Durham, NH 03824
Regionalization within the glandular epithelium of araneid silk glands has been revealed by several authors using various experimental methods. Therelationshipof this regionalization to the potential complexity of silk is discussed. We also review studies on the regulation of secretory protein synthesis in araneid silk glands, with an unavoidable emphasis on the major ampullate glands, the most frequently studied spider silk glands. A brief discussion of the relationship between web composition and the nutritional requirements of araneid spiders follows. Lastly, we describe a mechanism which allows juvenile araneid spiders to produce ampullate fibers during proecdysis (the preparatory period before ecdysis), a time when the primary major and minor ampullate glands are being remodeled and are temporarily nonfunctional. Two sets of secondary major and minor ampullate glands function alternately in successive proecdyses. (Only one set is functional in each juvenile stadium.) Both sets are nonfunctional and atrophied in adults (i.e. after thefinalmolt). The silk glands of araneid spiders are of several types (Figure 1). Of these, the major ampullate glands have received the most attention. They give rise to nonsticky silk fibers which are used in draglines and various elements of orb webs, including mooring lines, framelines, temporary and hub spirals, and radii. The functions of fibers produced by the minor ampullate glands are uncertain. They commonly accompany major ampullatefibers,especially in radii (i,2), and may serve to reinforce the elements in which they occur. However, Work (2) has argued against this interpretation. Certainly, their infrequent or seemingly random occurrence in some structural elements suggests that they are not critical to the functioning of such elements. To date, the use of minor ampullatefibers,without concurrent use of major ampullatefibers,has only been observed when bridging lines are being produced (5). This provides the first convincing evidence for a specific function for minor ampullatefibers.The flagelliform glands, which resemble the ampullate glands superficially, produce the corefibersof the orb web's adhesive spiral, while the aggregate glands produce the viscid, aqueous solution that envelops these corefibers(4,5). Pyriform glands giveriseto attachment disks, the cements that secure ampullatefibersto substrates, and probably also to the deposits that are used, e.g. during web construction, to cement intersecting ampullate
0097-6156/94/0544-0029$06.00/0 © 1994 American Chemical Society In Silk Polymers; Kaplan, D., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1993.
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fibers to one another (e.g. 7,2,5-7). The aciniform glands are involved in various activities, including swathing prey (along with fibers from pyriform and ampullate glands) (e.g. 5,7,8), producing 'molting threads' at ecdysis, and, probably at least in some species, eggcase construction. The principal fibers used in constructing the eggcase are products of the cylindrical (=tubuliform) glands, which are present only in females (e.g. 9-77). Spiders of the subfamily Nephilinae, as well as spiders in the genus Zygiella, were formerly classified as members of the family Araneidae (see 72), but are presently included in the family Tetragnathidae (see 75). (Both families are included in the superfamily Araneoidea.) Nephila clavipes, a member of the Nephilinae, has been and is often used in research on silk glands and their products. In this chapter, devoted primarily to discussing araneid silk glands, we have not hesitated to include the results of research performed using species in either of these transferred taxa. Where mention is made of other non-araneid spiders, the family to which the spiders belong is stated. Otherwise, it can be assumed that particular species referred to are members of the family Araneidae. Regionalization in Silk Glands From a gross morphological perspective, regionalization is immediately apparent in all silk glands in the form of a clear distinction between the gland's duct and the secretory epithelium of the body of the gland. The latter synthesizes and secretes into the gland's lumen proteins (in general) destined for extracorporeal use, while the former provides, in part, a conduit for passage of luminal contents to the spider's exterior. The duct is also undoubtedly involved in the final, critical processing of the material passing through its lumen and has been the subject of detailed ultrastructural studies in several types of silk glands from Araneus diadematus and Nephila clavata (14-22). In the major ampullate and minor ampullate glands of araneids, and to a lesser extent in the flagelliform glands, two morphological regions composing the body of the gland are recognized, the ampulla and the tail. The distinction between the two is essentially based on width, the tail being a relatively narrow tube and the ampulla a sac with an expanded lumen in which the secretory products of the epithelial cells are amassed (see Figure 4). In manytypesof silk glands different and well segregated cell types can also be distinguished in the body of a gland (reviewed in 23,24). In araneids, the secretory epithelia of minor ampullate, flagelliform and pyriform glands are composed of two, sharply delineated regions (cell types) which exocytose histochemically and ultrastructurally distinct secretory granules into the lumen, while major ampullate glands contain either two or three regions, depending on the genus (23-25). The border between ampulla and tail is not coincident with the border between cell types. For example, in the major ampullate glands of Araneus and Argiope, about three-fourths of the ampulla, that portion closest to the junction with the duct and furthest from the tail, is composed of one cell type while the remainder of the ampulla and the entire tail are composed of a second cell type (26). It has been observed that uniformity, with respect to secretory granules and other cellular components (e.g. glycogen particles, microtubules, Golgi apparatus, microvilli), does not necessarily exist even within one region (14,26). In the ampulla-specific cell type of the major ampullate glands of Argiope bruennichi, for example, gradual changes in both the size and histochemical affinities of the secretory granules are reportedly evident over the length of this region (26). With regard to the other cellular components mentioned, nonuniformity generally takes the form of gradual changes in abundance over the length of the region. The secretory epithelia of aggregate, cylindrical and aciniform glands are composed of only one cell type in araneids (23,24). Nevertheless, regionalization of a sort may occur in such glands. Kovoor (26) has described certain secretory granules in the cylindrical glands of Zygiella x-notata which, based on histochemical evidence, are present only in the most distal third of the body of the gland. And it is evident just from
In Silk Polymers; Kaplan, D., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1993.
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dissecting gravid Argiope aurantia that their cylindrical glands are not uniform in all respects from end to end. Examining the length of the contorted body of one of these glands one encounters a dazzling array of colors, produced by the luminal contents, with segments of different color merging into one another. To our knowledge the nature of this phenomenon has not been investigated. In the aggregate glands of Araneus diadematus, Kovoor (26) has observed a narrow region in the body of the gland, occupying a position between the rest of the glandular body and the duct, which differs from the rest of the glandular epithelium. The cells in this region are narrower than elsewhere and histochemical tests reveal abundant glycogen deposits and granulesrichin protein, unlike the results obtained for the bulk of the glandular epithelium. Evidence from Fibers, Luminal Contents, Nucleic Acid Sequencing. The histochemical, histological and ultrastructural evidence available, not only from araneids but from a variety of representatives of the liphistiomorph, mygalomorph and araneomorph suborders (reviewed in 23,24, see also 22,27-33), argues strongly for many silks being composed of more than one protein. Evidence obtained by other methods from luminal contents, the silks themselves and, recently, from nucleic acid sequence data, lead to the same conclusion. In major ampullate silk fibers from several araneid species (as well as Nephila and Nephilengys) a sheath-core morphology was commonly observed after fibers had been supercontracted in water and then re-extended (34). Work and Young (35) have noted that this behavior certainly does not prove that sheath and core differ in chemical composition, but they cite the ease with which the two can be made to separate and the intraspecific and intraindividual variability in amino acid composition of major ampullate silk as evidence which suggests that such is the case. In silk glands of several mygalomorphs (24,36,37) and in the pyriform glands of A. diadematus (38) it has been reported that the products of the different cell types remain separate and distinct even within the lumen of the duct, givingrisetofiberswith sheathcore morphologies. If the same description applies to araneid ampullate glands, as Kovoor and Zylberberg (38) have indicated, then it is possible that the sheath and core observed by Work (34) are products of the ampulla-specific and tail cell types, respectively. Two reports have provided additional evidence that some silks have a substructure. Using transmission electron microscopy, structural heterogeneity has been observed in cylindrical, pyriform and aciniform type A fibers from two species in the family Uloboridae (39), as well as in cylindrical glandfibersfrom A. aurantia (40). Moreover, treatment of A. aurantia cylindrical glandfiberswith trypsin and a solution of urea, SDS and 2-mercaptoethanol revealed thesefibersto be composed of fibrils embedded within a matrix (40) (Figure 2). Even greater heterogeneity in the composition of cylindrical fibers from this species is indicated by the results of SDS-PAGE. When extracts made from eggcases and cylindrical gland luminal contents were electrophoresed more than ten proteins were resolved (40). That cylindrical gland fibers can be compositionally heterogeneous is not unexpected as it has been shown that two histochemically distinct types of secretion granules are synthesized by the (single cell type of) cylindrical glands in some species (23,24). In addition, TEM has revealed that individual secretory granules in the cylindrical glands of Nephila clavata can be heterogeneous (20). Finally, in regard to heterogeneity in silkfibers,sequence data from partial cDNA clones has provided evidence of two, and only two, proteins in major ampullate silk from Nephila clavipes (41-44). Phosphatases. High alkaline phosphatase activity was observed in the silk glands of certain insects and spiders more than forty years ago, with pronounced regional distributions reported in some glands (45,46). In Figure 3 we present data for the major ampullate gland alkaline and acid phosphatases of Araneus cavaticus, expressed in U/L. [One unit of alkaline or acid phosphatase activity is defined as that amount of enzyme which will hydrolyze 1 //mol of /?-nitrophenyl phosphate or cc-naphthyl phosphate,
In Silk Polymers; Kaplan, D., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1993.
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Figure 1. Diagrams showing the gross morphology and locations of the different silk gland types within the opisthosoma of an adult female Larinioides sclopetarius. Only the silk glands in one-half of the opisthosoma are shown. (A) Major ampullate (left),flagelliform(middle) and minor ampullate (right) glands. (B) Aggregate glands. (C) Cylindrical glands. (D) Clusters of aciniform glands opening on posterior median and posterior lateral spinnerets, and the more ventrally located cluster of pyriform glands opening on anterior lateral spinnerets. (Reproduced with permission from ref. 77. Copyright 1969 American Microscopical Society.)
Figure 2. Eggcase of Argiope aurantia treated with bovine pancreatic trypsin (Sigma Chemical, T-8253) for 2 h at room temperature,rinsedthree times with distilled water and prepared for SEM as described previously (101). In Silk Polymers; Kaplan, D., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1993.
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400
0
ACP
ALP
ACP
ALP
Figure 3. Acid and alkaline phosphatases (ACP and ALP, respectively) of the ampulla-specific and tail cell types of the major ampullate silk glands of adult female Araneus cavaticus. Glands were severed very near the junction between the two cell types, within the ampulla-specific cell type, resulting in tail homogenates that contained a very small amount of the ampulla-specific cell type while ampulla homogenates were free of the tail cell type. The junction between the two cell types occurs about three-quarters of the way into the ampulla, when approaching from the direction of the duct (on right). Both cell types were homogenized in 200 piL distilled water. Acid phosphatases were assayed by the addition of 20 /
