BIOLOGY 468/568

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PRINCIPLES AND APPLICATIONS OF ELECTRON MICROSCOPY

STUDENT NAME:

Adam Gemar

PROJECT TITLE:

Ultrastructural and Morphological Variations in the Tail, Sac, and Duct of the Ampullate Silk Gland in Araneus diadematus (Araneidae)

Species Identification:

• Kingdom: Animalia
  
  
• Phylum: Arthropoda
  
  
• Class: Chelicerata
  
  
• Order: Araneae
  
  
• Family: Araneidae
  
  
• Genus: Araneus
  
  
• Species: diadematus

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Abstract

The ampullate silk gland of the spider, Araneus diadematus, produces proteinaceous fibers (silk) for draglines and the scaffolding of the web. The ultrastructure of the various regions of the silk gland are discribed by eletron microscopy. The tail harbors approximately 90% of the protein synthesis within the silk gland. Silk is storaged for use in the sac of the ampullate gland. The ducts function maybe the orientation of the silk fibers and water absorption. Large accumations of rough endoplasmic reticulum and mitochondria are observed in the tail. While at least four different protein droplets are found is the sac. These droplets can be readily seen emptying into the lumenal cavity. The duct contained large nuclei, some of which held nuclear inclusion bodies.

Key Words:

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INTRODUCTION

Silk is produced by various animals at different life stages for a variety of purposes. Caterpillars, for example, produced silk from modified salivary glands, and is spun from a spinneret near the mouth. The silk from ant-lions is produced by modified Malpighian tubes and is spun from the caudal end of the alimentary canal. However, the silk glands of spiders are the most complicated known (Comstock, 1948; Savory, 1928). They are located in the ventral side of the abdominal cavity and the silk is spun from spinnerets through spigot at the rear (Figure 1 and 2).

In spiders seven different silk glands have been recognized, but no spider has been found to possess all seven kinds. Most spiders, however, possess at least three silk glands; the aciniform glands, the pyriform glands, and the ampullaceal glands (Brusca, 1990). All the glands produce silk for different functions, and it seems that each gland synthesizes and secretes different types of silk for the various functions (Beckwitt, 1994; Brusca, 1990; Candelas, 1981). Silks from different glands have been shown to differ in their amino acid composition and physical properties (Beckwitt, 1994).

The ampullaceal (ampullate) silk gland of the spiders in the genus Araneus diadematus (Araneidea) has its particular function of rapid production of fibroin. This silk is used for the scaffolding ofthe web and the dragline trailed by the spider (Bell, 1969; Comstock, 1965; Candelas, 1981; Lombardi, 1990). The ampullate gland can be divided into three different parts; the duct, the ampulla (sac), and the tail. As described in the literature (Bell, 1969; Candelas, 1981; Lombardi, 1990) the tail is the site of 90% of the ampullate gland's protein synthetic activity, the ampulla is a storage site for the protein, and the duct is involved in secreting and ordering the silk.


The purpose of this study is to examine the ultrastructure and morphology of the three distinct regions of the ampullate silk gland in Araneus diadematus via electron microscopy.

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MATERIALS AND METHODS

Adult female Araneus diadematus spiders were collected in Long Beach, California and placed individually (to prevent cannibalism) in small glass containers. The small size of the containers discourages web-building. All the spiders were feed on house flies two days before the study began.

Dissection

The opisthosoma (abdomen) was removed from the prosoma by cutting through the pedicel. Once the abdomen was removed, the silk glands were dissected in a glutaraldehyde-phosphate fixative by cutting the cuticle from the pedicel opening to the spinnerets around the sides of the body. The exposed glands were excised with much care to avoid fragmenting the tissue. This was difficult, however, because of their small size and the stickiness of the glands.

TEM Preparation

The glands were fixed by immersion in 3.0% glutaraldehyde Sorensen phosphate buffer at pH 7.4 for one hour (Bozzola, 1992). Followed by two 30 minutes Sorensen phosphate buffer washes. The fixed glands were post-fixed in 1% osmium tetroxide aqueous solution for 1 hour on ice. The glands were dehydrated in a graded series of ethanols (30%, 50%, 70%, 95%, and 100%), and transferred to two 10 minute propylene oxide washes and embedded in Spurr resin (Bozzola, 1992).

Gold sections (ultrathins) were cut on a mechanical Sorvall MT-2 microtome with glass knives and mounted on uncoated copper grids (200 bars/cm). The sections were stained with uranyl acetate and lead citrate and examined with a Jeol-1200 EXII transmission electron microscope.

SEM Preparation

For examination of the spinnerets, a live spider was directly immersed in boiling water (Coddingtion 1989). The spinnerets spread open by this method for direct viewing by SEM. The whole spider was then ultrasonicated, while in fixative for five minutes, to remove dirt and debris. Fixation and dissection for SEM examination was prepared as the above TEM preparations. After the dehydration process the specimens (whole spider and ampullate silk gland) were dried in a LADD Model 28000 critical point drier using liquid CO2 as the transitional fluid (critical temp 31.1oC, critical pressure 1.073 PSI). The dried specimens were then mounted on stubs and coated with gold/palladium in a Hummer I sputter coater. The spinneret and the silk glands were examined in a AMR 1000 scanning electron microscope.

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RESULTS

The ampullate gland of the spider Araneus diadematus can be divided, by its gross anatomy, into three distinct regions. The long, slender tail, which has convoluted U-turns (Figure 3). The tail leads to the wide portion of the gland, the sac (ampullate) (Figure 3). The sac then joins the duct (Figure 4), which is connected to the spigot of the spinneret.

The Tail

Cross sections through the tail region of the ampullate gland is largely comprised of rough endoplasmic reticulum. In certain sections of the tail dense accumulations of rough endoplasmic reticulum was found (Figure 5). Mitochondria were also scattered throughout the cytoplasm of the tail (Figure 6). Within the cytoplasm and among numerable membrane electron dense granules, probably glycogen, were present (Figure 6and 7). These granules appear to be strictly association with the membranous material. No golgi apparatus were visible in any of the five tails examined. Also, few to no protein droplets were noticed within these regions.

The Sac (ampullate)

The obvious structures noticed within the sac of the ampullate silk gland were four to five spherical droplets or bodies. These droplets can be differentiated from one another by their morphology (Figure 8 and 9 and 10). They can be differentiated from one another by the range of moderate to heavy electron- opacity. The droplets contain a granular material somewhat fiberous in appearance. At the surface of the lumen of the silk gland in appears that the droplets are being emptied into the lumen (Figure 9). Endoplastic reticulum and mitochodrai are scarce within the sac, but the ER is found as a few strains meandering in close proximity to the spherical protein droplets.

The Duct

The duct is comprised of three distinct layers in which at least two contain membranous material and mitochondria (Figure 11). The layers are separated by distinct basal membranes. The outer epithelium is somewhat constant in diameter (0.3 um), while the inner two vary at different regions around the duct. The layer laying above the lumen is electron-opaque and contains few droplets similar in consistency to the material in the lumen.

Nuclei are present in the more translucent layer of the duct and nuclear inclusion bodies are distinctively nocticeable (Figure 12). This is probable the gene for the silk protein. Interestly, this is the area is where major gene ampification occurs. However, the duct is not known for its synthesis of the silk protein.

Transmission Electron Microscopy







PLATE 1; Figures 1-2 :(SEM-spinneret and spigots)



PLATE 2; Figures 3-4 :(SEM-Silk gland:tail/sac and duct)



PLATE 3; Figures 5-6 :TEM-Rough Endoplasmic Reticulum and Mitochondria and Glycogen



PLATE 4: Figures 7-8: TEM-Membrane/Glycogen (Tail) and Protein Droplets (Sac)





PLATE 5: Figures 9-10: TEM-Protein Emptying into Lumen and Variations of the Spherical Protein Droplets (Sac)




PLATE 6: Figure 11-12: TEM-The Three Distinct Layers and Nuclei/Nuclear Inclusion Bodies (Duct)

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DISCUSSION

The Tail

The majority of silk synthesis occurs in the tail portion of the gland, as state previously. While actively building weds, the rate of protein synthesis is about 10% of the gland weight (Bell, 1969; Candelas, 1981). To the great extent of rough endoplasmic reticulum found extensively throughout the tail, its appears that the tail is specialized for the production of rapid and large amounts of protein. On the basis of the number of mitochondria in the tail compared to the sac and the duct, the tail would be much more highly energetic than the latter two. However, in terms of protein productivity, one would expect that Golgi would be present. It is highly probable that Golgi was not detected because of the few numbers of glands sectioned. Or possibly a different post©fixative is needed to be incorporated, such as reduced©osmium. Similarly to this study, Bell and Peakall (1969) was unable to locate any Golgi while working with the spider Araneus sericatus. A study involving the spider Nephila clavipes by Plazaola and Candelas (1991), they described Golgi complex within the tail region. Upon examination of Plazaola and Candelas tail micrographs, the organelle they designate of Golgi complex have "non-typical" features within the cells and were not noticeable.

The Sac

The sac is the site for storage of protein synthesized by the tail. The numerable spherical droplets within the sac seem to be consistent with this account of a storage site and the droplets are waiting to be used. The function and purpose of the different electron-opacities droplets is not known. Probably the spherical droplets are of different protein properties and densities.

The Duct

The exact function of the duct is not known, however, speculations are that of silk is orientation into fibers in the duct. The silk protein in the sac is an alpha conformation, while in the silk fibers, the protein is a beta-pleated sheet (Bell, 1969). Water absorption maybe another function of the duct. Experiments show that the water content of silk in the sac is higher then that silk leaving the duct (Witt).

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Acknowledgements

I would like to thank Todd Chapman for being a brother and Brigdet Zepp for being a sister. Dr. Thomas Douglass for leading me in the right directions. Above all, I would like to thank Dr. Zed Mason for instruction and above all knowledge on the electron microscopes. This project would have not succeeded without my fuzzy, eight legged friends.

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CITATIONS

Beckwitt, Richard. and Steve Arcidiacono. (1994) Sequence conservation in the C-terminal region of spider silk proteins (Spidroin) from Nephila clavipes (Tetragnathidae) and Araneus bicentenarius (Araneidae). March 4. p.6661-6663.

Bell, Allen L., and David B. Peakall. (1969) Changes in fine structure during silk protein production in the ampullate gland of the spider, Araneus sericatus. The Journal of Cell Biology. 42:284-295.

Brusca, Richard C., and Gary J. Brusca. (1990) Invertebrates. Sinauer Associates, Inc. Sunderland, Massachusetts. p. 506-509.

Candelas, G.C., and A. Plazaloa. (1991) Stimulation of fibroin synthesis elicits ultrastructural modifications in spider silk secretory cells. Tissue and Cell. 23:(2)277-284.

Candelas, Graciela C., and Jose Cintron. (1981) A spider fibroin and its synthesis. The Journal of Experimental Zoology. 216:1-6.

Coddington, Jonathan A. (1989) Spinneret silk spigot morphology: Evidence for the monophyly of orbweaving spiders, Cyrtophorinae (Araneidae). and the group Theridiidae plus Nesticidae. Journal of Arachnology. 7:(1)71-95.

Comstock, John Henry. (1965). The Spider Book. Cornell University Press. Ithaca, New York.

Lombardi, Stephen J., and David L. Kaplan. (1990) The amino acid composition of major ampullate gland silk (dragline) of Nephila clavipes (Araneae, Tatragnathidae). Journal of Arachnology. 8:(3) 297-306.

Savory, Theodore H. (1928). The Biology of Spiders. Sidgwick and Jackson, LTD. London. p. 40-43; 71-76

Witt, P. N., C. F. Reed, and D.B. Peakall. A Spider's Web Problems in Regulatory Biology. Springer Verlag OHG., Berlin. In Press.

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List of Abbreviations Used