Arthropod Structure & Development xxx (2015) 1e9
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The embryonic origin of the ampullate silk glands of the spider Cupiennius salei Maarten Hilbrant a, b, *, Wim G.M. Damen a, c a
Institute for Genetics, University of Cologne, Zülpicher Straße 47a, 50674 Cologne, Germany Institute for Developmental Biology, University of Cologne, Zülpicher Straße 47b, 50674 Cologne, Germany c Department of Genetics, Friedrich Schiller University, Jena, Philosophenweg 12, 07743 Jena, Germany b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 14 February 2015 Received in revised form 31 March 2015 Accepted 1 April 2015 Available online xxx
Silk production in spiders is considered a key innovation, and to have been vital for the diversification of the clade. The evolutionary origin of the organs involved in spider silk production, however, and in particular of the silk glands, is poorly understood. Homologies have been proposed between these and other glands found in arachnids, but lacking knowledge of the embryonic development of spider silk glands hampers an evaluation of hypotheses. This study focuses on the embryonic origin of the largest silk glands of the spider Cupiennius salei, the major and minor ampullate glands. We show how the ampullate glands originate from ectodermal invaginations on the embryonic spinneret limb buds, in relation to morphogenesis of these buds. Moreover, we visualize the subsequent growth of the ampullate glands in sections of the early postembryonic stages. The invaginations are shown to correlate with expression of the proneural gene CsASH2, which is remarkable since it has been proposed that spider silk glands and their nozzles originate from sensory bristles. Hence, by confirming the ectodermal origin of spider silk glands, and by describing the (post-)embryonic morphogenesis of the ampullate glands, this work provides a starting point for further investigating into the genetic program that underlies their development. © 2015 Elsevier Ltd. All rights reserved.
Keywords: Spider Silk Spinneret Ampullate gland Ectoderm Invagination
1. Introduction Spiders employ silk for a wide range of tasks, including burrowing, locomotion, protection, courtship behaviour and predation, for which different sorts of silk are required. Therefore depending on species, developmental stage and sex, a single spider can possess up to seven different types of silk glands (Apstein, 1889; Mullen, 1969; Richter, 1970). The glands excrete their products through cuticular nozzles that are typically placed on specialized appendages called spinnerets. Spiders exert fine control over the movements of their spinnerets, allowing for the construction of complex structures. Indeed, the structural and behavioural intricacy of the
Abbreviations: ASp, MSp, PSp, anterior spinneret, medial spinneret, posterior spinneret; iASp, iMSp, iPSp, invagination sites on ASp, MSp, and PSp; lbO4, lbO5, limb buds of opisthosomal segments 4 and 5; MaA, MiA, Major and Minor Ampullate Glands. * Corresponding author. Institute for Developmental Biology, University of Cologne, Zülpicher Straße 47b, 50674 Cologne, Germany. Tel.: þ49 (0) 221 470 3238. E-mail addresses: [email protected] (M. Hilbrant), wim.damen@uni-jena. de (W.G.M. Damen).
spider silk producing organs forms an evolutionary innovation that is unique in the animal kingdom, and the system is probably the most diagnostic apomorphy of spiders (Shear et al., 1989). However, despite their ecological importance (Foelix, 2011; Nentwig and Heimer, 1987; Starrett et al., 2012), and the role that the silk producing organs probably had in the diversification of both spiders and their prey (Vollrath and Selden, 2007), their evolutionary origin, and in particular that of the silk glands, remains poorly understood. The best data we currently have are on the nature of the spinnerets. Both classical studies, comparing the musculature of spinnerets and other spider appendages (Whitehead and Rempel, 1959), as well as more recent gene expression studies (Hilbrant, 2008; Pechmann and Prpic, 2009) support the long standing idea that spinnerets are serial homologs of the prosomal appendages, and that they evolved via modification of a developmental mechanism that repressed opisthosomal appendages in the lineage leading to spiders. However, the function of the spinnerets is closely integrated with the function of the silk glands, and it is not clear how this integration came about or how silk glands originated (reviewed by Shultz, 1987).
http://dx.doi.org/10.1016/j.asd.2015.04.001 1467-8039/© 2015 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Hilbrant, M., Damen, W.G.M., The embryonic origin of the ampullate silk glands of the spider Cupiennius salei, Arthropod Structure & Development (2015), http://dx.doi.org/10.1016/j.asd.2015.04.001
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Several hypotheses exist. For example, it has been proposed that spider silk glands might have their origin in accessory glands, similar to those associated with the gonads of female specimens of certain other arachnid orders (Pocock, 1895). Another gonad related theory is inspired by the occurrence of spermatophores (sperm capsules) in many arachnids, and suggests an evolutionary link between the glands that are used to make spermatophores and the silk glands that males of many spider species use to build a sperm web (Marples, 1967; Nentwig and Heimer, 1987). A further theory suggests that silk glands might derive from coxal glands. These glands are part of the osmoregulatory and excretory system of many arachnids, including spiders, and are associated with their appendages. An interesting implication of the coxal gland theory is that silk might have evolved from waste products (Savory, 1960). More recent ideas postulate that silk glands are derived from dermal glands, and that nozzles might have evolved from sensory hairs (Bond, 1994; Palmer, 1990). Additional data in support of the latter hypothesis comes from the discovery of fluids originating from cuticular structures at the tips of the walking legs of certain spiders (Gorb et al., 2006; Rind et al., 2011) and other arachnids (Peattie et al., 2011). One way to test these hypotheses is by comparing the morphological and molecular basis of silk gland and nozzle development with the development of possible homologous organs. In this study we therefore describe the early post-embryonic and embryonic development of the silk glands of the Central American wandering spider Cupiennnius salei. In particular, we focus on the major (MaA) and minor (MiA) ampullate glands, which are large glands involved in making resilient silk such as the draglines. We identify these glands in histological sections of the early postembryonic stages, the first instar and postembryo. Moreover, we trace them back to ectodermal invagination sites on the embryonic spinneret limb buds, and relate their appearance and growth to spinneret limb bud morphogenesis. Interestingly, some of the invagination sites correlate with expression of the proneural gene CsASH2. We discuss these results in the light of the recent proposal that spider silk glands and their cuticular nozzles might originate from sensory bristles. Our results provide the first detailed description of embryonic spinneret and silk gland development using modern tools, and are in concordance with the idea that spider silk glands evolved from ectodermal organs. 2. Material and methods 2.1. Maintenance and dissection of adult spiders Spiders were cultured as described previously (Prpic et al., 2008a). Adults were sacrificed by first placing them at 4 C for 1 h, followed by sedation with CO2 and by then halving the prosoma with a sharp blade. Adult silk glands and spinnerets were dissected in phosphate buffered saline (PBS). 2.2. Sections of first instar and postembryo Specimens were prepared for sectioning by fixing them for 2 days in the following solution: 1% (w/v) picric acid (Fluka) dissolved in 1,4-dioxan (Sigma), 85 vols.; 37% formaldehyde (Sigma), 10 vols.; concentrated formic acid (Fluka), 5 vols. Specimens were washed once for 8 h and once for two days in pure 1,4-dioxan to remove excess picric acid, then incubated for 12 h in a 1:1 mixture of fresh 1,4-dioxan and SPURR epoxy embedding (Serva Electophoresis, Modified SPURR Embedding Kit) and subsequently moved to 100% SPURR resin. After 6 h, specimens were moved to an embedding mould and incubated at 70 C for 18 h to harden the resin. Trimmed blocks were cut in 5 mm slices on a JUNG 2065 SUPERCUT
microtome using a glass blade. Sections were stained with a mixture of 1% azure II (Aldrich) and 1% methylene blue (according to Ehrlich, Fluka) in a 1% aqueous borax solution (Sigma Aldrich) as described in Pernstich et al. (2003). After staining, sections were mounted in fresh SPURR resin, covered and incubated overnight at 70 C, followed by brightfield imaging on a compound microscope. 2.3. Scanning electron microscopy Samples for SEM imaging were prepared as described previously (Wolff and Hilbrant, 2011). Images were made with a Hitachi S-3400N in BSE mode at 25e30 keV. 2.4. Whole mount in situ hybridization Embryo fixation and whole mount in situ hybridization was performed according to standard methods (Prpic et al., 2008b, c). The CsASH2 riboprobe was made using the same plasmid as published previously (Stollewerk et al., 2001). 2.5. Immunohistochemistry and phalloidin staining Embryos were dechorionated with bleach and directly transferred to a 4% paraformaldehyde solution, after which the vitellin membranes were ruptured with forceps, followed by gently rocking for 20 min at room temperature. Next, vitellin membranes were removed and embryos were blocked and permeabilized for 3 h with PBSþ0.3% Triton-X-100, supplemented with 5% normal goat serum. They were then incubated either in a 1:500 dilution of FITCconjugated phalloidin (Sigma P5282) at 4 C o/n for staining of the actin cytoskeleton, or, for combined antibody and phalloidin staining, first incubated with a 1:200 dilution of rat-anti-Prospero antibody (Weller and Tautz, 2003) at 4 C o/n, followed by intensive washing and incubation with Cy5-anti-Rat secondary antibody and FITC-conjugated phalloidin, both at a 1:500 dilution, at 4 C o/n (all dilutions in PBS-T/NGS). Embryos were then flat mounted in Prolong Gold Antifade Mountant (Molecular Probes), followed by confocal imaging. 2.6. Confocal imaging and 3D analysis Confocal laser scanning microscopy was performed using a Leica TCS SP2. 3D projections and rotations were made using Imaris 5.7.2 (Bitplane AG) in the Surpass View window. Clipping planes were used to box out volumes and to create virtual z-stack cross-section images. 3. Results 3.1. The adult spinnerets and silk glands of C. salei In order to trace the developmental origin of the silk glands, we first examined the silk producing organs of an adult C. salei female. The spinning field, consisting of anterior (ASp), medial (MSp) and posterior (PSp) spinnerets, is located at the posterior end of the opisthosoma (Fig. 1a, b). The ASps derive from the fourth opisthosomal segment (O4) and the MSps and PSps from the fifth opisthosomal segment (O5) (Pechmann et al., 2010; Wolff and Hilbrant, 2011). The opisthosomal segments posterior to the spinnerets (O6eO12) are greatly reduced and form the anal tubercle, bearing the anus (Fig. 1b) (Wolff and Hilbrant, 2011). The distal surfaces of the spinnerets are covered in nozzles, each of which connects to a silk gland. The four large nozzles of the ampullated glands stand out. These are found on the ASp (2, Fig. 1b’) and the MSp (2, Fig. 1b”).
Please cite this article in press as: Hilbrant, M., Damen, W.G.M., The embryonic origin of the ampullate silk glands of the spider Cupiennius salei, Arthropod Structure & Development (2015), http://dx.doi.org/10.1016/j.asd.2015.04.001
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Fig. 1. The silk producing organs of a C. salei adult female. a. Ventral view of the pro- (Pro) and opisthosoma (Opistho), illustrating the position and relative size of the spinning field (Spin). Scale bar: 1 cm. BL: book lung b. Dissected spinning field and anal tubercle (At) of the left body half. Scale bar: 1 mm. b’, b”. The anterior (ASp) and medial (MSp) spinnerets, but not the posterior spinnerets (PSp), bear two ampullate nozzles each (arrows). Scale bars: 100 mm. c. Dissected major (MaA) and minor (MiA) ampullate glands, as well as aciniform (Ac), piriform (Pir) and tubuliform (Tub) glands (the MaA glands from the right body half were lost in this preparation, whereas all four MiA glands are visible). Scale bar ¼ 5 mm. d. Schematic drawing of the approximate position of the silk glands in the ventral opisthosoma. Lateral view, glands shown for one body half. e. Schematic drawing of how the different silk gland types connect to the spinnerets (one body half), and subdivision of the ampullate glands into tails, sacs and ducts (drawing modified from McGregor et al., 2008).
Dissection reveals the gross morphology of the different types of glands (Fig. 1c). Largest and most conspicuous are the ampullate glands. Adult female spiders bear, per body half, two MaAs leading to the two large nozzles on the corresponding ASp, and two MiAs ending in the two large nozzles on the MSp. Both types of ampullate glands consist of at least three parts: a long duct, a sac and a tail (Fig. 1cee) (Bell and Peakall, 1969). In C. salei the sacs and tails of the MaAs are located in the anterior lateral parts of the opisthosoma, slightly posterior to the book lungs (Fig. 1a, d). The MiAs lie medially and closer to the spinnerets. Also in close vicinity to the spinnerets lies a mass of smaller glands, consisting of piriform, aciniform and tubuliform glands (Fig. 1c, d). Tubuliform glands, for the production of egg case proteins, are unique to females. Hence, they were not observed while dissecting an adult male spider. Another sexual dimorphism seen in adult males is the presence of only one MaA and MiA each per body half. 3.2. The spinnerets and silk glands of juvenile stages C. salei spiders become adult after ten to twelve molts (Melchers, 1963). During juvenile life, the mass and number of glands increases with each molt. The juvenile stages therefore have a reduced set of silk glands compared to the adults. The first silk is secreted by the first instar, which is the last stage that resides in the protective cocoon produced by the mother (Wolff and Hilbrant, 2011). The spinning field of the first instar bears the full complement of spinnerets and all of these have silk gland nozzles, as well as sensory hairs (Fig. 2a). The ASp bears two large and two smaller nozzles (Fig. 2b, arrows and arrowheads respectively). The former are likely the nozzles of the major ampullate glands, which is apparent from their bulbous base, their position on the spinnerets and the observation that dragline silk is originating from them (Fig. 2b, asterisks). Four and three nozzles are present on the MSp and PSp, respectively (Fig. 2c,d); from their morphology it is not evident what silk gland type they are associated with. Fig. 2eeh depict sagittal sections of the opisthosoma of a 24 day old first instar. In lateral sections from one opisthosoma half, two glandular sacs were observed directly posterior to the book lung.
Based on their position, shape and size, they were identified as MaA sacs (arrows in Fig. 2e, e’, f, f’). More medial sections show ventral muscle nodes (Fig. 2g, g’) (Peters, 1967). Close to these muscle nodes lie two smaller sacs, just below and anterior to the cloaca (arrows in Fig. 2h, h’). The morphology of these sacs is less clear, but it is plausible that they are MiA sacs. Other silk glands could not be unmistakably identified in these sections, but dense tissue inside the spinnerets might, partially, constitute silk glands (Fig. 2h). The postembryo (Fig. 3a) precedes the first instar stage (Wolff and Hilbrant, 2011). It is difficult to distinguish the individual spinnerets on the external surface at this stage. The cuticle covering the spinning field is shaped into several transversal grooves, lacking sensory hairs and nozzles (Fig. 3b). The ASps, MSps and PSps have, however, already developed as epidermal structures underneath the cuticle (Fig. 3ceh; 3d’eh’). Dense tissue is visible inside each of the spinnerets (Fig. 3feh). Two glandular sacs are associated with the ASp, one of which extends about 350 mm into the opisthosoma (arrows in Fig. 3deg; Fig. 3d’eg’). Other structures visible in sections of the ventral posterior of the postembryo include the tubular trachea and components of the digestive system, such as the Malphigian tubules and the cloaca, as well as the protruding anal tubercle. 3.3. Morphogenetic changes of the spinneret limb buds The limb buds of opisthosomal segments four and five (lbO4 and lbO5) are the primordia of the spinnerets (Fig. 4). During late embryonic development, their morphology changes considerably. At the start of inversion at stage 14 (Wolff and Hilbrant, 2011) lbO4 is slightly larger than lbO5, and both have a globular and undifferentiated appearance, with many enlarged mitotic cells on the surface e indicating tissue growth (Fig. 4a’). Ventral to the limb buds, the ventral neuro-ectoderm (VNE) is evident as point-like depressions (Fig. 4a’; arrowheads) (Stollewerk et al., 2001; Wolff and Hilbrant, 2011). At stage 15 both limb buds have increased in size and have elongated dorsal-ventrally (Fig. 4b). Moreover, the surface of the center of lbO4 has become slightly depressed (Fig. 4b; asterisk) and at the ventral border of lbO5 a pore becomes visible (Fig. 4b’; arrow). The central depression of lbO4 is more
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Fig. 2. Silk producing organs of the first instar. a-d. SEM micrographs. a. Overview of the spinning field, showing anterior (ASp), medial (MSp), posterior (PSp) spinnerets and anal tubercle (At). Scale bar, 100 mm. b. Detail of the ASp. Arrows indicate presumptive nozzles of the major ampullate glands excreting silk threads (asterisks), arrowheads mark the nozzles of further silk glands. Scale bar, 20 mm. c, d. Details of MSp and PSp, showing four and three nozzles respectively (arrowheads). Scale bars, 15 mm. eeh: Series of sagittal sections of one half of the opisthosoma from lateral (e) to medial (h), stained with azure II and methylene blue. Scale bars 200 mm. e’eh’: Interpretations of sections e-h. Yolk sacs are shown as light grey pockets. Muscle structures are depicted with orange. BL, book lung; Clo, cloaca; Col, colulus; Hrt, heart; MaA, major ampullate gland; Malp, Malpighian tubule; MiA, minor ampullate gland; Sep, septa between yolk sacs; t8 and t9, muscle nodes following Peters (1967).
pronounced at stage 16 (Fig. 4c) and here too, a pore appears (Fig. 4c’; arrow). Cells in this depression seem to be organized in a pattern encircling the pore (Fig. 4c’). Stage 17 marks dorsal closure and the end of inversion (Wolff and Hilbrant, 2011). At this stage the overall shapes of lbO4 and lbO5 diverge; unlike lbO4, lbO5 continues to elongate dorsal-ventrally and divides into two portions, the later medial and posterior spinnerets (Fig. 4d; double arrow head). The positions of the pores remain visible as shallow depressions on the future ASp and MSp during stages 17e19 (Fig. 4def; arrows). Meanwhile the dorsal and ventral portions of lbO5 separate further from each other, the spinneret primordia of both opisthosomal segments flatten and the configuration of anterior (ASp), medial (MSp) and posterior (PSp) spinnerets becomes apparent (Fig. 4e and f). During ventral closure of the opisthosoma (stage 20) spinnerets of both halves come together and form the spinning field (see Wolff and Hilbrant, 2011). 3.4. Invaginations on the spinneret limb buds 3D reconstruction of lbO4 and lbO5 using confocal microscopy stacks of DAPI stained embryos was used to obtain further insight
into the epithelial changes on the spinneret limb buds. This 3D reconstruction confirms that ectodermal invaginations correlate with the positions of the pores observed on ASp and MSp (Fig. 5aec; arrows). Staining of filamentous actin, which visualizes the constricted cell processes of invaginating cells (Stollewerk and Seyfarth, 2008; Stollewerk et al., 2001), revealed three separate sites (iASp, iMSp and iPSp) on the primordia of all three spinnerets (Fig. 5dei and supplementary movie S1) with multiple invaginations at each site. Supplementary video related to this article can be found at http://dx.doi.org/10.1016/j.asd.2015.04.001. These invaginations fall into two categories, based on size and growth dynamics of the actin bundles; shallow invaginations that stay at the surface of the epithelium, and invaginations that continue to extend into the limb bud as development proceeds. Shallow invaginations appear around stage 17, at all three invagination sites (Fig. 5fei). Long actin bundles were found at sites iASp and iMSp only, and correspond to the deep invaginations in Fig. 5aec. These appear at stage 15 and continuously elongate from stage 15e20, after which stage confocal imaging is no longer possible due to the forming cuticle (Fig. 5dei).
Please cite this article in press as: Hilbrant, M., Damen, W.G.M., The embryonic origin of the ampullate silk glands of the spider Cupiennius salei, Arthropod Structure & Development (2015), http://dx.doi.org/10.1016/j.asd.2015.04.001
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Fig. 3. Silk producing organs of the postembryo. a, b. SEM micrographs a. Ventral view, showing position and relative size of the spinning field (Spin). Scale bar ¼ 200 mm. b. Detail of the spinning field showing the locations of sagittal sections d-h and the approximate positions of the spinneret primordia of the left body half, underlying the cuticle. Scale bar ¼ 40 mm. c. Sagittal section of a whole postembryo. Dashed box indicates the location of d-h. Scale bar ¼ 500 mm. d-h. Sagittal sections of the posterior end of the opisthosoma, stained with azure II and methylene blue. Scale bars ¼ 100 mm. d’eh’. Interpretations of sections in d-h. Yolk sacs are shown in light grey. The blue line depicts the ectoderm, underlying the cuticle shown in black. Muscle fibres are drawn in orange. At, anal tubercle; ASp, anterior spinneret; Clo, cloaca; Hrt, heart; Malp, Malpighian tubule; MSp, medial spinneret; Opistho, opisthosoma; Pro, prosoma; Proc, proctodeum; PSp, posterior spinneret; Sgl, presumptive silk gland; t10, t10 muscle node; Tsp, tracheal spiracle; Tt, tubular trachea.
Fig. 5d’ei’ depict concentrations of filamentous actin at iASP in more detail. They first appear at the surface at stage 15 (Fig. 5d’; arrow) and a single bundle extends to about 30 mm during stages 16e17 (Fig. 5e’,f’; arrow). At stages 17 and 18, smaller actin accumulations appear at the surface (Fig. 5f’,g’; arrowheads). By stage 19, one of these small actin bundles has extended further into the limb bud (Fig. 5h’; double arrowhead) and elongates to about 20 mm at stage 20 (Fig. 5i’). Simultaneously the initial large bundle has extended about 60 mm into the limb bud (Fig. 5i’; arrow).
3.5. Nervous system markers on the spinneret limb buds To distinguish between invaginations of the silk glands and developing neural tissue, we used two markers, CsASH2 and Prospero. These markers are expressed in the developing peripheral nervous system (Stollewerk and Seyfarth, 2008; Weller and Tautz, 2003). Transcripts of CsASH2, a spider homolog of the insect proneural achaeteescute genes, are expressed in a broad domain on lbO4 (co-localizing with iASp), at the ventral margin of lbO5 (colocalizing with iMSp), but not at the dorsal portion of lbO5 (Fig. 6a).
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Fig. 4. SEM micrographs showing morphogenesis of the embryonic spinneret limb buds. a. Ventral-lateral overview of a stage 14 embryo, indicating the fourth prosomal leg (L4) and limb buds of opisthosomal segments O2eO5 (lbO2-lbO5). Scale bar ¼ 200 mm. a’ef. SEM micrographs of lbO4 and lbO5 at stages 14e19. a’, b’ and c’ show details (dashed boxes) from a, b and c, respectively. Scale bars ¼ 25 mm. Arrowheads, invagination sites of ventral neuroectoderm; asterisk, slight depression on lbO4; arrows, pores on lbO4 and lbO5; double arrowheads, groove separating lbO5 into MSp and PSp.
In addition, CsASH2 is expressed in invaginations of the neuroectoderm, as described previously (Stollewerk et al., 2001). We did not detect expression of Prospero protein on either lbO4 or lbO5 (Fig. 6b), but could detect Prospero expression in the neuroectoderm. 4. Discussion 4.1. Embryonic ectodermal origin of the ampullate silk glands In several studies, it has been stated that spider silk glands derive ~ os-Cuevas, 1995), although from the ectoderm (e.g. Kovoor and Mun a detailed description of the developmental origin of spider silk glands is still missing. Such description, however, is needed in order to test hypotheses about the evolutionary origin of the spider silk producing organs. Therefore, we describe here the development of external and internal structure of the silk producing organs of the adult, the first instar, the postembryo and embryonic stages of the wandering spider C. salei, with a particular focus on the ampullate silk glands (MaA and MiA). We have traced their origins starting in the adult and following it back via the first instar and the postembryo to embryonic stages, and postulate that they originate as ectodermal invaginations on the spinneret primordia. In adult C. salei spiders, the sacs of the MaA lie in the anterior of the opisthosoma; the sacs of the MiA lie slightly more posterior. Both connect, via long ducts, to large nozzles on the spinnerets. In the first instar, we observed MaA sacs approximately half way along the anterioreposterior axis of the opisthosoma, while in the preceding stage, the postembryo, developing glandular structures lie closely to the spinnerets and no glands were found further anteriorly in the opisthosoma. These results indicate that the ampullate glands originate during embryonic development in close proximity of the spinnerets.
In concordance, we identified invagination sites (iASp, iMSp and iPSp) on the ectoderm of the spinneret limb buds during mid to late embryogenesis. The positions of these invagination sites on the spinneret primordia correspond to the positions of the nozzles on the spinnerets of the first instar, and suggest that at least a subset of the ectodermal invaginations of the embryonic spinnerets constitute primordia of ampullate silk glands. An alternative explanation for these ectodermal invaginations is that they represent precursor cells of sensory organs. Indeed, mechanoreceptors have been described on the spinnerets of adult C. salei spiders, near the nozzles of the major ampullate glands (Gorb and Barth, 1996). Moreover, CsASH2, a gene that has been associated with sensory precursor development on the walking legs (Stollewerk and Seyfarth, 2008), is expressed in the area of iASp and iMSp, which would be in support of this possibility. On the other hand, however, Prospero, another marker for sensory precursor groups (Stollewerk and Seyfarth, 2008; Weller and Tautz, 2003), is not found at any of the spinneret invagination sites at stage 16. Also, the morphology and growth dynamics of the large invaginations at the CsASH2 positive area of iASp and iMSp neither resemble the small nor the large sensory precursor groups that are present on the prosomal appendages (Stollewerk and Seyfarth, 2008). One way of combining these seemingly contradictory results is to postulate that some of the invaginations on the embryonic spinnerets constitute silk gland primordia, and some sensory precursor groups. Alternatively, CsASH2 might be involved in the development of the silk glands and/or the cuticular nozzles at iAPS and iMSP, rather than in sensory organ development. In any case, further work is needed to distinguish between the fates of the different developing structures on the spinneret primordia. Finally, the origin of ampullate glands at the spinneret ectoderm implies remarkable growth and differentiation of these glands
Please cite this article in press as: Hilbrant, M., Damen, W.G.M., The embryonic origin of the ampullate silk glands of the spider Cupiennius salei, Arthropod Structure & Development (2015), http://dx.doi.org/10.1016/j.asd.2015.04.001
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Fig. 5. Invagination sites on the spinneret limb buds. a. lbO4 and lbO5 of a stage 17 embryo, visualised by 3D projection of a confocal stack of a flat-mounted embryo stained with the nuclear dye DAPI. Scale bar 25 mm. a’. Same as a, additionally showing the projection of virtual z-stack cross-sections corresponding to b and c. b, c. Virtual sections through lbO4 and lbO5, respectively. Scale bars ¼ 25 mm. dei. Actin cytoskeleton of lbO4 and lbO5 visualised using FITC-conjugated phalloidin in a series of embryonic stages. Scale bars 50 mm. d’ei’. Projection of a boxed out volume surrounding invagination site iASp. Bright FITC-phalloidin staining shows the formation of invaginating pockets (arrowheads) and subsequent extension of the invagination. Arrows and double arrowheads indicate the tips of invaginations. Scale bars 25 mm. At, anal tubercle; ect, ectoderm; mes, mesoderm; VNE, ventral neuroectoderm.
Fig. 6. Nervous system markers in the spinneret primordia a. In situ hybridisation showing that CsASH2 (dark blue/purple) is expressed surrounding the iASP on lbO4 and the iMSP on lbO5 (arrowheads), as well as in the VNE, in a stage 15 embryo. DAPI nuclear counter staining in bright blue. b. Antibody staining against C. salei Prospero (red) shows Prospero positive cells in the VNE, but not on lbO4 and lbO5. FITC phalloidin was used to detect filamentous actin (green). Scale bars 50 mm. iASp and iMSP, large invaginations sites on lbO4 and lbO5; lbO4, lbO5, limb buds of opisthosomal segments O4 and O5 respectively; VNE, ventral neuroectoderm.
through the opisthosoma, analogous to the silk glands of Bombyx mori (Julien et al., 2004), which raises several developmental questions. Which are the molecular factors involved in growth and sub-compartmentalization of these glands? Are there internal structures that direct the growth? One possibility is that the ampullate glands follow the ventral musculature of the opisthosoma, since we detected a close spatial association between these structures. Another possibility is that the ampullate silk glands grow along the septa that line the yolk sacs (Rempel, 1957).
4.2. The developmental origin of the smaller silk gland types In this study, we focus on the development of the ampullate glands. Ampullate glands are found in many different spider lineages (Boutry et al., 2011) and the tensile strength of their product, the dragline silk, is renowned (Rising, 2007). However, the evolution of different types of glands and silk proteins, for example for the production of attachment discs, has truly revolutionized the complexity of the spider silk producing system and its versatile
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ecological use (Blackledge, 2006; Craig, 2003; Vollrath and Selden, 2007). Therefore, in order to understand these innovations, the evolution and development of the smaller (e.g. aciniform, piriform) and more specialized (e.g. tubuliform) glands should be considered too. A handful of smaller nozzles are present on the spinnerets of the first instar, including the PSp that bear no ampullate glands (Fig. 2bed) and it is therefore likely that the associated glands develop during embryonic development. Potential silk gland invagination sites on the embryonic primordia of the PSp (Fig. 5), which is the spinneret that does not bear ampullate glands, support this hypothesis. However, the number of these smaller glands increases with each molt, and mature tubuliform glands are found only in adult females, both indicating that C. salei silk gland development is, to a large extent, postembryonic. Future studies on the tissue underlying the spinnerets in older juvenile stages are needed to compare embryonic and postembryonic silk gland development. 4.3. The evolution of spider silk producing organs In a series of recent articles it was debated whether present-day tarantulas produce silk at the tips of their walking legs (Foelix et al., 2013 and references therein) and although it was convincingly shown that the latter theory is erroneous, these studies led to the discovery of non-silk fluids that originate from the adhesive pads of rez-Miles and spiders and other arachnids (Peattie et al., 2011; Pe Ortiz-Villatoro, 2012). The nature of these fluids is not clear, but they appear to originate from chemosensory hairs (Foelix et al., 2012). Regardless of the function and nature of these fluids in present-day tarantulas, the finding of a secretory function of these hairs suggest an intriguing scenario in which spider silk glands and their nozzles evolved via transformation of chemoreceptor hairs, as has been proposed previously (Palmer, 1990). The absence of silk gland nozzles on the legs of tarantulas makes it less likely for this process to have taken place on the walking appendages, but the genetics underlying the development of secretory sensory hairs could have been co-opted elsewhere on the body of a common ancestor of spiders. The data presented here not only confirm the ectodermal origin of spider silk glands. Expression of the neuronal marker gene CsASH2 on the ASp and MSp spinneret primordia also highlights the need to characterize, on spinnerets, the expression and function of candidate genes known to be involved in development of sensory organs. Of particular interest is the potential role of these genes in the embryonic and postembryonic development of spider nozzles and silk glands. Acknowledgements We would like to thank C. Wolff for his help with SEM imaging and for kindly providing two images (Fig. 3a,b), K. A. Panfilio for help with silk gland dissections, A. Stollewerk for providing the Prospero antibody and Oxford Brookes University (UK) for use of SEM facilities. M. Pechmann, K. A. Panfilio and two anonymous reviewers provided helpful comments on the manuscript. This project was funded by the European Union via the Marie Curie RTN grant “ZOONET” (MRTN-CT-2004-005624) to WGMD. References Apstein, C., 1889. Bau und Funktion der Spinndrüsen der Araneida. Arach. Naturgesch. 55, 29e74. Bell, A.L., Peakall, D.B., 1969. Changes in fine structure during silk protein production in the ampullate gland of the spider Araneus sericatus. J. Cell. Biol. 42, 284e295.
Blackledge, T.A., 2006. Silken toolkits: biomechanics of silk fibers spun by the orb web spider Argiope argentata (Fabricius 1775). J. Exp. Biol. 209, 2452e2461. Bond, J., 1994. Seta-spigot homology and silk production in first instar Antrodiaetus unicolor spiderlings (araneae: antrodiaetidae). J. Arachn. 19e22. a Boutry, C., Rez c, M., Blackledge, T.A., 2011. Plasticity in major ampullate silk production in relation to spider phylogeny and ecology. PLoS One 6, e22467. Craig, C.L., 2003. Spiderwebs and Silk, Tracing Evolution from Molecules to Genes to Phenotypes. Oxford University Press, New York. Foelix, R., Erb, B., Rast, B., 2013. Alleged silk spigots on tarantula feet: electron microscopy reveals sensory innervation, no silk. Arthropod Struct. Dev. 42, 209e217. Foelix, R.F., 2011. Biology of Spiders. Oxford University Press, Oxford. Foelix, R.F., Rast, B., Peattie, A.M., 2012. Silk secretion from tarantula feet revisited: alleged spigots are probably chemoreceptors. J. Exp. Biol. 215, 1084e1089. Gorb, S., Barth, F., 1996. A new mechanosensory organ on the anterior spinnerets of the spider Cupiennius salei (Araneae, Ctenidae). Zoomorphology 116, 7e14. €tsch, W., Walther, P., Gorb, S.N., Niederegger, S., Hayashi, C.Y., Summers, A.P., Vo 2006. Biomaterials: silk-like secretion from tarantula feet. Nature 443, 407. Hilbrant, M., 2008. Development and Evolution of the Spider Silk Producing System. PhD thesis. University of Cologne, Cologne, pp. 1e156. Julien, E., Coulon-Bublex, M., Garel, A., Royer, C., Chavancy, G., Prudhomme, J., Couble, P., 2004. Silk gland development and regulation of silk protein genes. In: Gilbert, L., Iatrou, K., Gill, S. (Eds.), Comprehensive Molecular Insect Science. Pergamon Press, Oxford, pp. 369e384. ~ os-Cuevas, A., 1995. Embryonic and postembryonic morphogenesis Kovoor, J., Mun of the visual, venom- and silk-gland systems in two species of Peucetia (Araneae: Oxyopidae). Eur. J. Entomol. 92, 565e571. Marples, B.J., 1967. The spinnerets and epiandrous glands of spiders. J. Linn. Soc. Lond. 46, 209e222. McGregor, A.P., Hilbrant, M., Pechmann, M., Schwager, E.E., Prpic, N.-M., Damen, W.G., 2008. Cupiennius salei and Achaearanea tepidariorum: spider models for investigating evolution and development. BioEssays 30, 487e498. Melchers, M., 1963. Zur Biologie und zum Verhalten von Cupiennius salei (Keyserling), einer amerikanischen Ctenide. Zool. Jb Syst. 91, 1e90. Mullen, G.R., 1969. Morphology and histology of the silk glands in Araneus sericatus Cl. Trans. Am. Microsc. Soc. 88, 232e240. Nentwig, W., Heimer, S., 1987. Ecological aspects of spider webs. In: Nentwig, W. (Ed.), Ecophysiology of Spiders. Springer Berlin Heidelberg, Berlin, Heidelberg, pp. 211e225. Palmer, J.M., 1990. Comparative Morphology of the External Silk Production Apparatus of “Primitive” Spiders. Harvard University, Cambridge, Massachusetts. Peattie, A.M., Dirks, J.-H., Henriques, S., Federle, W., 2011. Arachnids secrete a fluid over their adhesive pads. PLoS One 6, e20485. Pechmann, M., Khadjeh, S., Sprenger, F., Prpic-Schaeper, N.-M., 2010. Patterning mechanisms and morphological diversity of spider appendages and their importance for spider evolution. Arthropod Struct. Dev. 39, 453e467. Pechmann, M., Prpic, N.-M., 2009. Appendage patterning in the South American bird spider Acanthoscurria geniculata (Araneae: Mygalomorphae). Dev. Genes. Evol. 219, 189e198. rez-Miles, F., Ortiz-Villatoro, D., 2012. Tarantulas do not shoot silk from their legs: Pe experimental evidence in four species of New World tarantulas. J. Exp. Biol. 215, 1749e1752. Pernstich, A., Krenn, H.W., Pass, G., 2003. Preparation of serial sections of arthropods using 2,2-dimethoxypropane dehydration and epoxy resin embedding under vacuum. Biotech. Histochem. 78, 5e9. Peters, R., 1967. Vergleichende Untersuchungen über Bau und Funktion der Spinnwarzen und Spinnwarzenmuskulatur einiger Araneen. Zool. Beitr. 13, 29e119. Pocock, R.I., 1895. Some suggestions on the origin and evolution of web-spinning in spiders. Nature 51, 417e420. Prpic, N.-M., Schoppmeier, M., Damen, W.G.M., 2008a. The American wandering spider Cupiennius salei. Cold Spring Harb. Protoc. pdb.emo103. Prpic, N.-M., Schoppmeier, M., Damen, W.G.M., 2008b. Collection and fixation of spider embryos. Cold Spring Harb. Protoc. Prpic, N.-M., Schoppmeier, M., Damen, W.G.M., 2008c. Whole-mount in situ hybridization of spider embryos. Cold Spring Harb. Protoc. Rempel, J.G., 1957. The embryology of the black widow spider, Latrodectus mactans (Fabr.). Canad. J. Zool. 35, 35e74. Richter, C., 1970. Morphology and function of the spinning apparatus of the Wolf spider Pardosa amentata (Cl.) (Araneae, Lycosidae). Z Morph Tiere 68, 37e68. Rind, F.C., Birkett, C.L., Duncan, B.-J.A., Ranken, A.J., 2011. Tarantulas cling to smooth vertical surfaces by secreting silk from their feet. J. Exp. Biol. 214, 1874e1879. Rising, A., 2007. Spider Dragline Silk e Molecular Properties and Recombinant Expression. PhD thesis. Swedish University of Agricultural Sciences, Uppsala, pp. 1e52. Savory, T.H., 1960. Spider webs. Sci. Am. 202, 114e124. Shear, W., Palmer, J., Coddington, J., Bonamo, P., 1989. A devonian spinneret e early evidence of spiders and silk use. Science 246, 479e481. Shultz, J.W., 1987. The origin of the spinning apparatus in spiders. Biol. Rev. Cam. Philos. Soc. 62, 89e113. Starrett, J., Garb, J.E., Kuelbs, A., Azubuike, U.O., Hayashi, C.Y., 2012. Early events in the evolution of spider silk genes. PLoS One 7, e38084. Stollewerk, A., Seyfarth, E.-A., 2008. Evolutionary changes in sensory precursor formation in arthropods: embryonic development of leg sensilla in the spider Cupiennius salei. Dev. Biol. 313, 659e673.
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The embryonic origin of the ampullate silk glands of the spider Cupiennius salei Maarten Hilbrant a, b, *, Wim G.M. Damen a, c a
Institute for Genetics, University of Cologne, Zülpicher Straße 47a, 50674 Cologne, Germany Institute for Developmental Biology, University of Cologne, Zülpicher Straße 47b, 50674 Cologne, Germany c Department of Genetics, Friedrich Schiller University, Jena, Philosophenweg 12, 07743 Jena, Germany b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 14 February 2015 Received in revised form 31 March 2015 Accepted 1 April 2015 Available online xxx
Silk production in spiders is considered a key innovation, and to have been vital for the diversification of the clade. The evolutionary origin of the organs involved in spider silk production, however, and in particular of the silk glands, is poorly understood. Homologies have been proposed between these and other glands found in arachnids, but lacking knowledge of the embryonic development of spider silk glands hampers an evaluation of hypotheses. This study focuses on the embryonic origin of the largest silk glands of the spider Cupiennius salei, the major and minor ampullate glands. We show how the ampullate glands originate from ectodermal invaginations on the embryonic spinneret limb buds, in relation to morphogenesis of these buds. Moreover, we visualize the subsequent growth of the ampullate glands in sections of the early postembryonic stages. The invaginations are shown to correlate with expression of the proneural gene CsASH2, which is remarkable since it has been proposed that spider silk glands and their nozzles originate from sensory bristles. Hence, by confirming the ectodermal origin of spider silk glands, and by describing the (post-)embryonic morphogenesis of the ampullate glands, this work provides a starting point for further investigating into the genetic program that underlies their development. © 2015 Elsevier Ltd. All rights reserved.
Keywords: Spider Silk Spinneret Ampullate gland Ectoderm Invagination
1. Introduction Spiders employ silk for a wide range of tasks, including burrowing, locomotion, protection, courtship behaviour and predation, for which different sorts of silk are required. Therefore depending on species, developmental stage and sex, a single spider can possess up to seven different types of silk glands (Apstein, 1889; Mullen, 1969; Richter, 1970). The glands excrete their products through cuticular nozzles that are typically placed on specialized appendages called spinnerets. Spiders exert fine control over the movements of their spinnerets, allowing for the construction of complex structures. Indeed, the structural and behavioural intricacy of the
Abbreviations: ASp, MSp, PSp, anterior spinneret, medial spinneret, posterior spinneret; iASp, iMSp, iPSp, invagination sites on ASp, MSp, and PSp; lbO4, lbO5, limb buds of opisthosomal segments 4 and 5; MaA, MiA, Major and Minor Ampullate Glands. * Corresponding author. Institute for Developmental Biology, University of Cologne, Zülpicher Straße 47b, 50674 Cologne, Germany. Tel.: þ49 (0) 221 470 3238. E-mail addresses: [email protected] (M. Hilbrant), wim.damen@uni-jena. de (W.G.M. Damen).
spider silk producing organs forms an evolutionary innovation that is unique in the animal kingdom, and the system is probably the most diagnostic apomorphy of spiders (Shear et al., 1989). However, despite their ecological importance (Foelix, 2011; Nentwig and Heimer, 1987; Starrett et al., 2012), and the role that the silk producing organs probably had in the diversification of both spiders and their prey (Vollrath and Selden, 2007), their evolutionary origin, and in particular that of the silk glands, remains poorly understood. The best data we currently have are on the nature of the spinnerets. Both classical studies, comparing the musculature of spinnerets and other spider appendages (Whitehead and Rempel, 1959), as well as more recent gene expression studies (Hilbrant, 2008; Pechmann and Prpic, 2009) support the long standing idea that spinnerets are serial homologs of the prosomal appendages, and that they evolved via modification of a developmental mechanism that repressed opisthosomal appendages in the lineage leading to spiders. However, the function of the spinnerets is closely integrated with the function of the silk glands, and it is not clear how this integration came about or how silk glands originated (reviewed by Shultz, 1987).
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Several hypotheses exist. For example, it has been proposed that spider silk glands might have their origin in accessory glands, similar to those associated with the gonads of female specimens of certain other arachnid orders (Pocock, 1895). Another gonad related theory is inspired by the occurrence of spermatophores (sperm capsules) in many arachnids, and suggests an evolutionary link between the glands that are used to make spermatophores and the silk glands that males of many spider species use to build a sperm web (Marples, 1967; Nentwig and Heimer, 1987). A further theory suggests that silk glands might derive from coxal glands. These glands are part of the osmoregulatory and excretory system of many arachnids, including spiders, and are associated with their appendages. An interesting implication of the coxal gland theory is that silk might have evolved from waste products (Savory, 1960). More recent ideas postulate that silk glands are derived from dermal glands, and that nozzles might have evolved from sensory hairs (Bond, 1994; Palmer, 1990). Additional data in support of the latter hypothesis comes from the discovery of fluids originating from cuticular structures at the tips of the walking legs of certain spiders (Gorb et al., 2006; Rind et al., 2011) and other arachnids (Peattie et al., 2011). One way to test these hypotheses is by comparing the morphological and molecular basis of silk gland and nozzle development with the development of possible homologous organs. In this study we therefore describe the early post-embryonic and embryonic development of the silk glands of the Central American wandering spider Cupiennnius salei. In particular, we focus on the major (MaA) and minor (MiA) ampullate glands, which are large glands involved in making resilient silk such as the draglines. We identify these glands in histological sections of the early postembryonic stages, the first instar and postembryo. Moreover, we trace them back to ectodermal invagination sites on the embryonic spinneret limb buds, and relate their appearance and growth to spinneret limb bud morphogenesis. Interestingly, some of the invagination sites correlate with expression of the proneural gene CsASH2. We discuss these results in the light of the recent proposal that spider silk glands and their cuticular nozzles might originate from sensory bristles. Our results provide the first detailed description of embryonic spinneret and silk gland development using modern tools, and are in concordance with the idea that spider silk glands evolved from ectodermal organs. 2. Material and methods 2.1. Maintenance and dissection of adult spiders Spiders were cultured as described previously (Prpic et al., 2008a). Adults were sacrificed by first placing them at 4 C for 1 h, followed by sedation with CO2 and by then halving the prosoma with a sharp blade. Adult silk glands and spinnerets were dissected in phosphate buffered saline (PBS). 2.2. Sections of first instar and postembryo Specimens were prepared for sectioning by fixing them for 2 days in the following solution: 1% (w/v) picric acid (Fluka) dissolved in 1,4-dioxan (Sigma), 85 vols.; 37% formaldehyde (Sigma), 10 vols.; concentrated formic acid (Fluka), 5 vols. Specimens were washed once for 8 h and once for two days in pure 1,4-dioxan to remove excess picric acid, then incubated for 12 h in a 1:1 mixture of fresh 1,4-dioxan and SPURR epoxy embedding (Serva Electophoresis, Modified SPURR Embedding Kit) and subsequently moved to 100% SPURR resin. After 6 h, specimens were moved to an embedding mould and incubated at 70 C for 18 h to harden the resin. Trimmed blocks were cut in 5 mm slices on a JUNG 2065 SUPERCUT
microtome using a glass blade. Sections were stained with a mixture of 1% azure II (Aldrich) and 1% methylene blue (according to Ehrlich, Fluka) in a 1% aqueous borax solution (Sigma Aldrich) as described in Pernstich et al. (2003). After staining, sections were mounted in fresh SPURR resin, covered and incubated overnight at 70 C, followed by brightfield imaging on a compound microscope. 2.3. Scanning electron microscopy Samples for SEM imaging were prepared as described previously (Wolff and Hilbrant, 2011). Images were made with a Hitachi S-3400N in BSE mode at 25e30 keV. 2.4. Whole mount in situ hybridization Embryo fixation and whole mount in situ hybridization was performed according to standard methods (Prpic et al., 2008b, c). The CsASH2 riboprobe was made using the same plasmid as published previously (Stollewerk et al., 2001). 2.5. Immunohistochemistry and phalloidin staining Embryos were dechorionated with bleach and directly transferred to a 4% paraformaldehyde solution, after which the vitellin membranes were ruptured with forceps, followed by gently rocking for 20 min at room temperature. Next, vitellin membranes were removed and embryos were blocked and permeabilized for 3 h with PBSþ0.3% Triton-X-100, supplemented with 5% normal goat serum. They were then incubated either in a 1:500 dilution of FITCconjugated phalloidin (Sigma P5282) at 4 C o/n for staining of the actin cytoskeleton, or, for combined antibody and phalloidin staining, first incubated with a 1:200 dilution of rat-anti-Prospero antibody (Weller and Tautz, 2003) at 4 C o/n, followed by intensive washing and incubation with Cy5-anti-Rat secondary antibody and FITC-conjugated phalloidin, both at a 1:500 dilution, at 4 C o/n (all dilutions in PBS-T/NGS). Embryos were then flat mounted in Prolong Gold Antifade Mountant (Molecular Probes), followed by confocal imaging. 2.6. Confocal imaging and 3D analysis Confocal laser scanning microscopy was performed using a Leica TCS SP2. 3D projections and rotations were made using Imaris 5.7.2 (Bitplane AG) in the Surpass View window. Clipping planes were used to box out volumes and to create virtual z-stack cross-section images. 3. Results 3.1. The adult spinnerets and silk glands of C. salei In order to trace the developmental origin of the silk glands, we first examined the silk producing organs of an adult C. salei female. The spinning field, consisting of anterior (ASp), medial (MSp) and posterior (PSp) spinnerets, is located at the posterior end of the opisthosoma (Fig. 1a, b). The ASps derive from the fourth opisthosomal segment (O4) and the MSps and PSps from the fifth opisthosomal segment (O5) (Pechmann et al., 2010; Wolff and Hilbrant, 2011). The opisthosomal segments posterior to the spinnerets (O6eO12) are greatly reduced and form the anal tubercle, bearing the anus (Fig. 1b) (Wolff and Hilbrant, 2011). The distal surfaces of the spinnerets are covered in nozzles, each of which connects to a silk gland. The four large nozzles of the ampullated glands stand out. These are found on the ASp (2, Fig. 1b’) and the MSp (2, Fig. 1b”).
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Fig. 1. The silk producing organs of a C. salei adult female. a. Ventral view of the pro- (Pro) and opisthosoma (Opistho), illustrating the position and relative size of the spinning field (Spin). Scale bar: 1 cm. BL: book lung b. Dissected spinning field and anal tubercle (At) of the left body half. Scale bar: 1 mm. b’, b”. The anterior (ASp) and medial (MSp) spinnerets, but not the posterior spinnerets (PSp), bear two ampullate nozzles each (arrows). Scale bars: 100 mm. c. Dissected major (MaA) and minor (MiA) ampullate glands, as well as aciniform (Ac), piriform (Pir) and tubuliform (Tub) glands (the MaA glands from the right body half were lost in this preparation, whereas all four MiA glands are visible). Scale bar ¼ 5 mm. d. Schematic drawing of the approximate position of the silk glands in the ventral opisthosoma. Lateral view, glands shown for one body half. e. Schematic drawing of how the different silk gland types connect to the spinnerets (one body half), and subdivision of the ampullate glands into tails, sacs and ducts (drawing modified from McGregor et al., 2008).
Dissection reveals the gross morphology of the different types of glands (Fig. 1c). Largest and most conspicuous are the ampullate glands. Adult female spiders bear, per body half, two MaAs leading to the two large nozzles on the corresponding ASp, and two MiAs ending in the two large nozzles on the MSp. Both types of ampullate glands consist of at least three parts: a long duct, a sac and a tail (Fig. 1cee) (Bell and Peakall, 1969). In C. salei the sacs and tails of the MaAs are located in the anterior lateral parts of the opisthosoma, slightly posterior to the book lungs (Fig. 1a, d). The MiAs lie medially and closer to the spinnerets. Also in close vicinity to the spinnerets lies a mass of smaller glands, consisting of piriform, aciniform and tubuliform glands (Fig. 1c, d). Tubuliform glands, for the production of egg case proteins, are unique to females. Hence, they were not observed while dissecting an adult male spider. Another sexual dimorphism seen in adult males is the presence of only one MaA and MiA each per body half. 3.2. The spinnerets and silk glands of juvenile stages C. salei spiders become adult after ten to twelve molts (Melchers, 1963). During juvenile life, the mass and number of glands increases with each molt. The juvenile stages therefore have a reduced set of silk glands compared to the adults. The first silk is secreted by the first instar, which is the last stage that resides in the protective cocoon produced by the mother (Wolff and Hilbrant, 2011). The spinning field of the first instar bears the full complement of spinnerets and all of these have silk gland nozzles, as well as sensory hairs (Fig. 2a). The ASp bears two large and two smaller nozzles (Fig. 2b, arrows and arrowheads respectively). The former are likely the nozzles of the major ampullate glands, which is apparent from their bulbous base, their position on the spinnerets and the observation that dragline silk is originating from them (Fig. 2b, asterisks). Four and three nozzles are present on the MSp and PSp, respectively (Fig. 2c,d); from their morphology it is not evident what silk gland type they are associated with. Fig. 2eeh depict sagittal sections of the opisthosoma of a 24 day old first instar. In lateral sections from one opisthosoma half, two glandular sacs were observed directly posterior to the book lung.
Based on their position, shape and size, they were identified as MaA sacs (arrows in Fig. 2e, e’, f, f’). More medial sections show ventral muscle nodes (Fig. 2g, g’) (Peters, 1967). Close to these muscle nodes lie two smaller sacs, just below and anterior to the cloaca (arrows in Fig. 2h, h’). The morphology of these sacs is less clear, but it is plausible that they are MiA sacs. Other silk glands could not be unmistakably identified in these sections, but dense tissue inside the spinnerets might, partially, constitute silk glands (Fig. 2h). The postembryo (Fig. 3a) precedes the first instar stage (Wolff and Hilbrant, 2011). It is difficult to distinguish the individual spinnerets on the external surface at this stage. The cuticle covering the spinning field is shaped into several transversal grooves, lacking sensory hairs and nozzles (Fig. 3b). The ASps, MSps and PSps have, however, already developed as epidermal structures underneath the cuticle (Fig. 3ceh; 3d’eh’). Dense tissue is visible inside each of the spinnerets (Fig. 3feh). Two glandular sacs are associated with the ASp, one of which extends about 350 mm into the opisthosoma (arrows in Fig. 3deg; Fig. 3d’eg’). Other structures visible in sections of the ventral posterior of the postembryo include the tubular trachea and components of the digestive system, such as the Malphigian tubules and the cloaca, as well as the protruding anal tubercle. 3.3. Morphogenetic changes of the spinneret limb buds The limb buds of opisthosomal segments four and five (lbO4 and lbO5) are the primordia of the spinnerets (Fig. 4). During late embryonic development, their morphology changes considerably. At the start of inversion at stage 14 (Wolff and Hilbrant, 2011) lbO4 is slightly larger than lbO5, and both have a globular and undifferentiated appearance, with many enlarged mitotic cells on the surface e indicating tissue growth (Fig. 4a’). Ventral to the limb buds, the ventral neuro-ectoderm (VNE) is evident as point-like depressions (Fig. 4a’; arrowheads) (Stollewerk et al., 2001; Wolff and Hilbrant, 2011). At stage 15 both limb buds have increased in size and have elongated dorsal-ventrally (Fig. 4b). Moreover, the surface of the center of lbO4 has become slightly depressed (Fig. 4b; asterisk) and at the ventral border of lbO5 a pore becomes visible (Fig. 4b’; arrow). The central depression of lbO4 is more
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Fig. 2. Silk producing organs of the first instar. a-d. SEM micrographs. a. Overview of the spinning field, showing anterior (ASp), medial (MSp), posterior (PSp) spinnerets and anal tubercle (At). Scale bar, 100 mm. b. Detail of the ASp. Arrows indicate presumptive nozzles of the major ampullate glands excreting silk threads (asterisks), arrowheads mark the nozzles of further silk glands. Scale bar, 20 mm. c, d. Details of MSp and PSp, showing four and three nozzles respectively (arrowheads). Scale bars, 15 mm. eeh: Series of sagittal sections of one half of the opisthosoma from lateral (e) to medial (h), stained with azure II and methylene blue. Scale bars 200 mm. e’eh’: Interpretations of sections e-h. Yolk sacs are shown as light grey pockets. Muscle structures are depicted with orange. BL, book lung; Clo, cloaca; Col, colulus; Hrt, heart; MaA, major ampullate gland; Malp, Malpighian tubule; MiA, minor ampullate gland; Sep, septa between yolk sacs; t8 and t9, muscle nodes following Peters (1967).
pronounced at stage 16 (Fig. 4c) and here too, a pore appears (Fig. 4c’; arrow). Cells in this depression seem to be organized in a pattern encircling the pore (Fig. 4c’). Stage 17 marks dorsal closure and the end of inversion (Wolff and Hilbrant, 2011). At this stage the overall shapes of lbO4 and lbO5 diverge; unlike lbO4, lbO5 continues to elongate dorsal-ventrally and divides into two portions, the later medial and posterior spinnerets (Fig. 4d; double arrow head). The positions of the pores remain visible as shallow depressions on the future ASp and MSp during stages 17e19 (Fig. 4def; arrows). Meanwhile the dorsal and ventral portions of lbO5 separate further from each other, the spinneret primordia of both opisthosomal segments flatten and the configuration of anterior (ASp), medial (MSp) and posterior (PSp) spinnerets becomes apparent (Fig. 4e and f). During ventral closure of the opisthosoma (stage 20) spinnerets of both halves come together and form the spinning field (see Wolff and Hilbrant, 2011). 3.4. Invaginations on the spinneret limb buds 3D reconstruction of lbO4 and lbO5 using confocal microscopy stacks of DAPI stained embryos was used to obtain further insight
into the epithelial changes on the spinneret limb buds. This 3D reconstruction confirms that ectodermal invaginations correlate with the positions of the pores observed on ASp and MSp (Fig. 5aec; arrows). Staining of filamentous actin, which visualizes the constricted cell processes of invaginating cells (Stollewerk and Seyfarth, 2008; Stollewerk et al., 2001), revealed three separate sites (iASp, iMSp and iPSp) on the primordia of all three spinnerets (Fig. 5dei and supplementary movie S1) with multiple invaginations at each site. Supplementary video related to this article can be found at http://dx.doi.org/10.1016/j.asd.2015.04.001. These invaginations fall into two categories, based on size and growth dynamics of the actin bundles; shallow invaginations that stay at the surface of the epithelium, and invaginations that continue to extend into the limb bud as development proceeds. Shallow invaginations appear around stage 17, at all three invagination sites (Fig. 5fei). Long actin bundles were found at sites iASp and iMSp only, and correspond to the deep invaginations in Fig. 5aec. These appear at stage 15 and continuously elongate from stage 15e20, after which stage confocal imaging is no longer possible due to the forming cuticle (Fig. 5dei).
Please cite this article in press as: Hilbrant, M., Damen, W.G.M., The embryonic origin of the ampullate silk glands of the spider Cupiennius salei, Arthropod Structure & Development (2015), http://dx.doi.org/10.1016/j.asd.2015.04.001
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Fig. 3. Silk producing organs of the postembryo. a, b. SEM micrographs a. Ventral view, showing position and relative size of the spinning field (Spin). Scale bar ¼ 200 mm. b. Detail of the spinning field showing the locations of sagittal sections d-h and the approximate positions of the spinneret primordia of the left body half, underlying the cuticle. Scale bar ¼ 40 mm. c. Sagittal section of a whole postembryo. Dashed box indicates the location of d-h. Scale bar ¼ 500 mm. d-h. Sagittal sections of the posterior end of the opisthosoma, stained with azure II and methylene blue. Scale bars ¼ 100 mm. d’eh’. Interpretations of sections in d-h. Yolk sacs are shown in light grey. The blue line depicts the ectoderm, underlying the cuticle shown in black. Muscle fibres are drawn in orange. At, anal tubercle; ASp, anterior spinneret; Clo, cloaca; Hrt, heart; Malp, Malpighian tubule; MSp, medial spinneret; Opistho, opisthosoma; Pro, prosoma; Proc, proctodeum; PSp, posterior spinneret; Sgl, presumptive silk gland; t10, t10 muscle node; Tsp, tracheal spiracle; Tt, tubular trachea.
Fig. 5d’ei’ depict concentrations of filamentous actin at iASP in more detail. They first appear at the surface at stage 15 (Fig. 5d’; arrow) and a single bundle extends to about 30 mm during stages 16e17 (Fig. 5e’,f’; arrow). At stages 17 and 18, smaller actin accumulations appear at the surface (Fig. 5f’,g’; arrowheads). By stage 19, one of these small actin bundles has extended further into the limb bud (Fig. 5h’; double arrowhead) and elongates to about 20 mm at stage 20 (Fig. 5i’). Simultaneously the initial large bundle has extended about 60 mm into the limb bud (Fig. 5i’; arrow).
3.5. Nervous system markers on the spinneret limb buds To distinguish between invaginations of the silk glands and developing neural tissue, we used two markers, CsASH2 and Prospero. These markers are expressed in the developing peripheral nervous system (Stollewerk and Seyfarth, 2008; Weller and Tautz, 2003). Transcripts of CsASH2, a spider homolog of the insect proneural achaeteescute genes, are expressed in a broad domain on lbO4 (co-localizing with iASp), at the ventral margin of lbO5 (colocalizing with iMSp), but not at the dorsal portion of lbO5 (Fig. 6a).
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Fig. 4. SEM micrographs showing morphogenesis of the embryonic spinneret limb buds. a. Ventral-lateral overview of a stage 14 embryo, indicating the fourth prosomal leg (L4) and limb buds of opisthosomal segments O2eO5 (lbO2-lbO5). Scale bar ¼ 200 mm. a’ef. SEM micrographs of lbO4 and lbO5 at stages 14e19. a’, b’ and c’ show details (dashed boxes) from a, b and c, respectively. Scale bars ¼ 25 mm. Arrowheads, invagination sites of ventral neuroectoderm; asterisk, slight depression on lbO4; arrows, pores on lbO4 and lbO5; double arrowheads, groove separating lbO5 into MSp and PSp.
In addition, CsASH2 is expressed in invaginations of the neuroectoderm, as described previously (Stollewerk et al., 2001). We did not detect expression of Prospero protein on either lbO4 or lbO5 (Fig. 6b), but could detect Prospero expression in the neuroectoderm. 4. Discussion 4.1. Embryonic ectodermal origin of the ampullate silk glands In several studies, it has been stated that spider silk glands derive ~ os-Cuevas, 1995), although from the ectoderm (e.g. Kovoor and Mun a detailed description of the developmental origin of spider silk glands is still missing. Such description, however, is needed in order to test hypotheses about the evolutionary origin of the spider silk producing organs. Therefore, we describe here the development of external and internal structure of the silk producing organs of the adult, the first instar, the postembryo and embryonic stages of the wandering spider C. salei, with a particular focus on the ampullate silk glands (MaA and MiA). We have traced their origins starting in the adult and following it back via the first instar and the postembryo to embryonic stages, and postulate that they originate as ectodermal invaginations on the spinneret primordia. In adult C. salei spiders, the sacs of the MaA lie in the anterior of the opisthosoma; the sacs of the MiA lie slightly more posterior. Both connect, via long ducts, to large nozzles on the spinnerets. In the first instar, we observed MaA sacs approximately half way along the anterioreposterior axis of the opisthosoma, while in the preceding stage, the postembryo, developing glandular structures lie closely to the spinnerets and no glands were found further anteriorly in the opisthosoma. These results indicate that the ampullate glands originate during embryonic development in close proximity of the spinnerets.
In concordance, we identified invagination sites (iASp, iMSp and iPSp) on the ectoderm of the spinneret limb buds during mid to late embryogenesis. The positions of these invagination sites on the spinneret primordia correspond to the positions of the nozzles on the spinnerets of the first instar, and suggest that at least a subset of the ectodermal invaginations of the embryonic spinnerets constitute primordia of ampullate silk glands. An alternative explanation for these ectodermal invaginations is that they represent precursor cells of sensory organs. Indeed, mechanoreceptors have been described on the spinnerets of adult C. salei spiders, near the nozzles of the major ampullate glands (Gorb and Barth, 1996). Moreover, CsASH2, a gene that has been associated with sensory precursor development on the walking legs (Stollewerk and Seyfarth, 2008), is expressed in the area of iASp and iMSp, which would be in support of this possibility. On the other hand, however, Prospero, another marker for sensory precursor groups (Stollewerk and Seyfarth, 2008; Weller and Tautz, 2003), is not found at any of the spinneret invagination sites at stage 16. Also, the morphology and growth dynamics of the large invaginations at the CsASH2 positive area of iASp and iMSp neither resemble the small nor the large sensory precursor groups that are present on the prosomal appendages (Stollewerk and Seyfarth, 2008). One way of combining these seemingly contradictory results is to postulate that some of the invaginations on the embryonic spinnerets constitute silk gland primordia, and some sensory precursor groups. Alternatively, CsASH2 might be involved in the development of the silk glands and/or the cuticular nozzles at iAPS and iMSP, rather than in sensory organ development. In any case, further work is needed to distinguish between the fates of the different developing structures on the spinneret primordia. Finally, the origin of ampullate glands at the spinneret ectoderm implies remarkable growth and differentiation of these glands
Please cite this article in press as: Hilbrant, M., Damen, W.G.M., The embryonic origin of the ampullate silk glands of the spider Cupiennius salei, Arthropod Structure & Development (2015), http://dx.doi.org/10.1016/j.asd.2015.04.001
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Fig. 5. Invagination sites on the spinneret limb buds. a. lbO4 and lbO5 of a stage 17 embryo, visualised by 3D projection of a confocal stack of a flat-mounted embryo stained with the nuclear dye DAPI. Scale bar 25 mm. a’. Same as a, additionally showing the projection of virtual z-stack cross-sections corresponding to b and c. b, c. Virtual sections through lbO4 and lbO5, respectively. Scale bars ¼ 25 mm. dei. Actin cytoskeleton of lbO4 and lbO5 visualised using FITC-conjugated phalloidin in a series of embryonic stages. Scale bars 50 mm. d’ei’. Projection of a boxed out volume surrounding invagination site iASp. Bright FITC-phalloidin staining shows the formation of invaginating pockets (arrowheads) and subsequent extension of the invagination. Arrows and double arrowheads indicate the tips of invaginations. Scale bars 25 mm. At, anal tubercle; ect, ectoderm; mes, mesoderm; VNE, ventral neuroectoderm.
Fig. 6. Nervous system markers in the spinneret primordia a. In situ hybridisation showing that CsASH2 (dark blue/purple) is expressed surrounding the iASP on lbO4 and the iMSP on lbO5 (arrowheads), as well as in the VNE, in a stage 15 embryo. DAPI nuclear counter staining in bright blue. b. Antibody staining against C. salei Prospero (red) shows Prospero positive cells in the VNE, but not on lbO4 and lbO5. FITC phalloidin was used to detect filamentous actin (green). Scale bars 50 mm. iASp and iMSP, large invaginations sites on lbO4 and lbO5; lbO4, lbO5, limb buds of opisthosomal segments O4 and O5 respectively; VNE, ventral neuroectoderm.
through the opisthosoma, analogous to the silk glands of Bombyx mori (Julien et al., 2004), which raises several developmental questions. Which are the molecular factors involved in growth and sub-compartmentalization of these glands? Are there internal structures that direct the growth? One possibility is that the ampullate glands follow the ventral musculature of the opisthosoma, since we detected a close spatial association between these structures. Another possibility is that the ampullate silk glands grow along the septa that line the yolk sacs (Rempel, 1957).
4.2. The developmental origin of the smaller silk gland types In this study, we focus on the development of the ampullate glands. Ampullate glands are found in many different spider lineages (Boutry et al., 2011) and the tensile strength of their product, the dragline silk, is renowned (Rising, 2007). However, the evolution of different types of glands and silk proteins, for example for the production of attachment discs, has truly revolutionized the complexity of the spider silk producing system and its versatile
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ecological use (Blackledge, 2006; Craig, 2003; Vollrath and Selden, 2007). Therefore, in order to understand these innovations, the evolution and development of the smaller (e.g. aciniform, piriform) and more specialized (e.g. tubuliform) glands should be considered too. A handful of smaller nozzles are present on the spinnerets of the first instar, including the PSp that bear no ampullate glands (Fig. 2bed) and it is therefore likely that the associated glands develop during embryonic development. Potential silk gland invagination sites on the embryonic primordia of the PSp (Fig. 5), which is the spinneret that does not bear ampullate glands, support this hypothesis. However, the number of these smaller glands increases with each molt, and mature tubuliform glands are found only in adult females, both indicating that C. salei silk gland development is, to a large extent, postembryonic. Future studies on the tissue underlying the spinnerets in older juvenile stages are needed to compare embryonic and postembryonic silk gland development. 4.3. The evolution of spider silk producing organs In a series of recent articles it was debated whether present-day tarantulas produce silk at the tips of their walking legs (Foelix et al., 2013 and references therein) and although it was convincingly shown that the latter theory is erroneous, these studies led to the discovery of non-silk fluids that originate from the adhesive pads of rez-Miles and spiders and other arachnids (Peattie et al., 2011; Pe Ortiz-Villatoro, 2012). The nature of these fluids is not clear, but they appear to originate from chemosensory hairs (Foelix et al., 2012). Regardless of the function and nature of these fluids in present-day tarantulas, the finding of a secretory function of these hairs suggest an intriguing scenario in which spider silk glands and their nozzles evolved via transformation of chemoreceptor hairs, as has been proposed previously (Palmer, 1990). The absence of silk gland nozzles on the legs of tarantulas makes it less likely for this process to have taken place on the walking appendages, but the genetics underlying the development of secretory sensory hairs could have been co-opted elsewhere on the body of a common ancestor of spiders. The data presented here not only confirm the ectodermal origin of spider silk glands. Expression of the neuronal marker gene CsASH2 on the ASp and MSp spinneret primordia also highlights the need to characterize, on spinnerets, the expression and function of candidate genes known to be involved in development of sensory organs. Of particular interest is the potential role of these genes in the embryonic and postembryonic development of spider nozzles and silk glands. Acknowledgements We would like to thank C. Wolff for his help with SEM imaging and for kindly providing two images (Fig. 3a,b), K. A. Panfilio for help with silk gland dissections, A. Stollewerk for providing the Prospero antibody and Oxford Brookes University (UK) for use of SEM facilities. M. Pechmann, K. A. Panfilio and two anonymous reviewers provided helpful comments on the manuscript. This project was funded by the European Union via the Marie Curie RTN grant “ZOONET” (MRTN-CT-2004-005624) to WGMD. References Apstein, C., 1889. Bau und Funktion der Spinndrüsen der Araneida. Arach. Naturgesch. 55, 29e74. Bell, A.L., Peakall, D.B., 1969. Changes in fine structure during silk protein production in the ampullate gland of the spider Araneus sericatus. J. Cell. Biol. 42, 284e295.
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