Abstract
Animal Cells and Systems 12: 109-116, 2008 Spinning Apparatus for the Dragline Silk in the Funnel- web Spider Agelena limbata (Araneae: Agelenidae) Jong-Gu Park and Myung-Jin Moon* Department of Biological Sciences & Institute of Basic Science, Dankook University , Cheonan 330-714, Korea Ab str a c t: Among the four kinds of silk glands in the dropping (Lucas, 1964; W ork, 1981, 1984; Tillinghast and funnel-web spider Agelena limbata, the ampullate gland for T o wnley , 1 987). dragline silk production is the most predominate one inIt is widely accepted that the spider silks, as one group of both sexes, and is composed of three functional parts - fibrous proteins with a single repetitive structure (Kaplan, excretory duct, storage ampulla and convoluted tail regions. 1998), are exceptional structural materials. Especially in Two pairs of major ampullate glands send secretory terms of strength, toughness and energy absorption prior to ductules to the anterior spinnerets, and another two pairs of break, the dragline silks are unmatched in the world of minor ampullate glands supply the middle spinnerets. synthetic or natural fibers (Gosline et al., 1984). By this There are no apparent differences between the major and reason, the dragline silks produced from the ampullate minor ampullate glands not only the external spigots butglands have received the most attention. also their internal silk glands. However, the microstructureAraneidae spiders use silk from the two kinds of ampullate is very unique in this spider, because each gland hasglands for web building (Coddington, 1986; Tillinghast and spherical shaped storage sac with twig-like branched tails. Townley, 1986, 1987). Cytologic aspects of spider silk Nevertheless, the wall of the secretory region is similarly elaboration have been studied previously in the case of the composed of a single layer of epithelial cells. The mature ampullate glands of Araneus (Peakall, 1965; Bell and secretory silks in glandular epithelium are closely packed Peakall, 1969; T illinghast and T ownley , 1986) and Nephila and accumulated as electron-opaque vesicles. Most of the (Tillinghast and Christenson, 1984; Moon and Tillinghast, secretory products which originated from the rough 2004). It has been noted in these reports that the two kinds endoplasmic reticula (rER) are grown up by fusion with the of ampullate glands (major and minor) produce different surrounding small vesicles however, the Golgi complex types of silk materials. However, exact functions of these does not seem to play an important role in this process of two kinds of silks for web building are still not known secretion.sufficiently. The funnel-web spider, also known as grass spider, is a Key words: fine structure, silk apparatus, funnel-web spider, group of spiders that make funnel-shaped webs, which they Agelena limbata use to trap insects. Systematically, they are close relatives of free-wandering wolf spiders (Coddington and Levi, 1991). Although they are among the most abundant and The silk glands of the araneid spiders produce four to nine conspicuous spiders in temperate areas, little is known kinds of fibers which are indifferently used for the about their spinning systems both of spinnerets and silk construction of their webs, retreats or burrows as well as glands. Moreover, the histological and fine structural egg cases or sperm webs (Coddington, 1986, Nentwig and properties of this gland have been nearly neglected except Heimer, 1987). Among the several types of silk glands in for the recent work of Park and Moon (2002), and several araneid spiders, the ampullate glands are mainly used to other brief studies (Kovoor, 1987). make dragline which maintains their weight in the case of Thus, we describe here the fine structural aspects of the ampullate silk gland, and production of secretory silk * T o whom correspondence should be addressed. within the glandular epithelium in the funnel-web spider, T el: +82-41-550-3445; Fax: +82-41-550-3409 E-mail: moonmj@dankook.ac.kr Agelena limbata, especially its functional morphology of ANIMAL CELLS AND SYSTEMS Vo l. 12 No. 2 109 Jong-Gu P ark and Myung-Jin Moon the spinning apparatus through light and scanning electron T okyo, Japan) field emission scanning electron microscop y microscopic observations.(FESEM) operated with accelerating voltages of 5-20 kV. MA TERIALS AND METHODS RE SU L T S Specimens of the funnel-web spiders Agelena limbata The spinnerets of the funnel-web spiders Agelena limbata (Araneae: Agelenidae) were collected in a local area near are composed of three pairs, and silk spigots are located at Cheonan campus of Dankook University , Cheonan, Korea. the surface of the spinnerets. The silk glands are connected All spiders were maintained under ambient conditions with with a particular type of spinning tubes, namely , ampullate, natural lighting in the special cages of wooden frames, and pyriform, aciniform and tubuliform (only in females) fed insects and water daily. Twelve specimens (6 females spigo ts. The ampu llate sp igots for dragline silks are located and 6 males) were anesthetized with CO and dissected on both the anterior and middle spinnerets (Fig. 1A). Two under a dissecting light microscope in a drop of spider pairs of major ampullate glands send secretory ductules to Ringer's solution consisting of 160 mmol/L NaCl, 7.5the anterior spinnerets (Fig. 1B,C), and another two pairs of mmol/L KCl, 4 mmol/L CaCl , 1 mmol/L MgCl , 4 mmol/minor ampullate glands supplies the middle spinnerets (Fig. 2 2 L NaHCO , 20 mmol/L glucose, pH 7.4 (Groome et al., 1D-F). When viewed from above the spinnerets, two large 1991). spinning tubes of ampullate spigots are the most prominent The specimens for histologic preparation were fixed in(Fig. 1C-D). buffered neutral formalin solution, embedded with In addition to the large spigots of the major ampullate Paraplast embedding medium (Polyscience Inc., USA) via glands, numerous small spigots of the pyriform glands are xylene, and stained with hematoxylin and eosin solution. also present at the anterior spinneret. Spigots of the For light microscopic observation, parts of the specimens pyriform are quite different from those of the ampullates were then dehydrated in ascending concentrations of with respect to size and morphology . The spinning tubes of ethanol and embedded in Poly/Bed 812-Araldite mixture the pyriform glands are only fo und on this anterior s p i n n e r e t (Polysciences Inc., USA) via propylene oxide. Longitudinal (Fig. 1B). Additional two pairs of minor ampullate spigots and transverse sections, 0.5-1.0 µ m thick, were obtained are connected on the median spinneret in both sexes (Fig. using a LKB-V Ultramicrotome. These semi-thin sections 1E). The average size of the minor ampullate spigot is were stained with 1% toluidine blue (dissolved in 1% so me what smaller than those of the major amp ullate spigots borax), and were photographed using Zeiss Axiophot on the anterior spinneret. All of these spigots are basically microscope coupled with Motic digital imaging system. composed of two main segments-more flexible basal For transmission electron microscopic examination, eachsegment and slender apical segment (Fig. 1F). silk gland was gently removed and fixed in the same The ampullate glands are the most prominent of all silk fixatives for pre- and p o st-fixation. Subsequ ently , the tissue glands observed in dissection. There are four pairs in this pieces were dehydrated in ascending concentrations of spider (Fig. 2A). Each is composed of a long excretory ethanol and embedded in Poly/Bed 812-Araldite mixture duct, the spherical storage sac and the convoluted tail. The (Polysciences Inc., USA) via propylene oxide. Ultra-thin microstructure of the storage sac is very unique in this sections were obtained from a LKB-V ultramicrotome, and spider, because they are spherical in shape instead of were double stained with uranyl acetate, followed by lead normal ampullar appearance (Fig. 2B,C). However, there citrate. The sections were examined with a JEM 100 CX-II are no apparent differences between the major and minor electron microscope (JEOL Ltd., Japan) at 80kV. ampullate glands. Characteristically, each ampullate gland For scanning electron microscopic examination, the has a spherical shaped storage sac with twig-like branched spinnerets were gently removed and fixed in a mixture oftails (Fig. 2D,E). 2% paraformaldehyde and 2.5% glutaraldehyde bufferedThe duct of the ampullate gland is basically composed of with 0.1 mol/L phosphate buffer at pH 7.4. Postfixation three superposed types of the layers which are inner cuticles, was done in 1% osmium tetroxide in the same buffer. monolayered epithelial cells and peripheral connective Following fixation, the specimens were washed several cells. The electron lucent subcuticles which have the times in 0.1 mol/L phosphate buffer and put through an functions of water removal and orientation of silk fibers ethanol series from 30 to l00% (30min at each concentration, during polymerization are well developed at the distal with one repeat at 100%). The specimens were transferred region of the duct ad jacen t to sp igot (Fig. 3A). Wh ereas, the to hexamethyldisilazane (HMDS) and were allowed to air endocuticles are developed at the rest of duct region, these dry . All samples were coated to a thickness of approximately subcuticles gradually disappeared at the middle and 20nm with gold-palladium alloy using a sputter coater, andproximal regions. Along the plane of transverse section, the they were examined on a Hitachi S-4300 (Hitachi Co., endocuticle shows two alternating light and dark bands 110 ANIMAL CELLS AND SYSTEMS Vol. 12 No. 2 Ampullate Silk in F unnel-web S pider Fig. 1. A: Scanning electron micrographs of the spinning apparatus in the female spider Agelena limbata . AS: anterior spinneret, MS: middle s p inneret , PS: pos ter i or s p inner et. B, C: Ant e r i or spi n ner et has 2 s p igot s of maj o r ampullat e gl a nds ( A m ) and numerous pyrif o rm spinning tubes. D-F: Middle spinneret has another two spigots of minor ampullate glands and a number of aciniform spigots. Scale bars indicate 500 µ m (A), 200 µ m (E), 100 µ m (B) and 50 µ m (C,D,F), respectively . (Fig. 3B,C). These granules after releasing from the epithelial cells The protein-synthesizing epithelial cells of ampullate frequently appear to aggregate with several others, forming silk gland is composed of tall columnar cells with large, amorphous electron dense deposits (Fig. 3E). These fine irregularly ellipsoidal nuclei. The secretory silks are granular materials, possibly proteins, seem to be transported synthesized within the glandular epithelial cells of the tail across the ap ical me mb rane by exocytotic activities, and ar e as secretory granules, and then released to the inner cavityadded to the luminal secretory products as precursors of the of the storage ampulla by the mechanism of the eccrineampullate silk (Fig. 3F). secretion (Fig. 3D). The mature secretory product in By transmission electron microscopic observation, inner glandular epithelium appears as almost spherical granules. cuticular intima which composed of three layers of cuticles ANIMAL CELLS AND SYSTEMS Vo l. 12 No. 2 111 Jong-Gu P ark and Myung-Jin Moon Fig. 2. Micrographs of internal silk glands in the female spider Agelena limbata . A-C: The ampullate glands (Am) are the most prominent of all silk glands observed in dissection. Each gland is composed of a long excretory duct, the spherical storage sac and the convolut ed tail. D-E: Each tail of the ampullate gland has some twig-like branching points similarly to that of simple branched tubular gland. Scale ba r s i n d i c a t e 2 m m (A) 1 mm (D,E) and 500 µ m (B,C), respectively . - subcuticle, endocuticle and exocuticle - are commonly are contain fine granulated material are found near the originated from the epithelial cells of the excretory duct nucleus (Fig. 4C). Numerous small vesicles, which (Fig. 4A). presumed to be the precursors of silk granules, also Several types of silk precursors appear at the secretory appeared at the base of this epithelium. Apparently, these region of the ampullate silk gland. These secretory silk vesicles are synthesized from the rER, and the secretory products accumulate in the luminal cytoplasm of the granules are formed by fusion with these small vesicles glandular epithelial cells as diverse forms of secretory(Fig. 4D). granules. Several types of secretory granules are seen in the The mature secretory product in glandular epithelium cytoplasm of the epithelium. The appearance of secretory appears as almost spherical granules. These electron dense granules differs from cell to cell and can be representative granules are densely packed and remain close to each other of the cellular maturation (Fig. 4B). without fusion (Fig. 4E). The luminal border of the cells In the cytoplasm of the glandular epithelial cell, broadcomprises a series of microvilli. The apices of the glandular cisternae of the rough endoplasmic reticulum (rER) which cells are fringed with short and irregular microvilli. The 112 ANIMAL CELLS AND SYSTEMS Vol. 12 No. 2 Ampullate Silk in F unnel-web S pider Fi g. 3. A-C : Light m i c r ographs of t h e exc r etor y duc t of the am pullat e gland wh ic h c o m pos ed of t h r ee c onc entr i c l a ye r s - i nner c u tic l es , columnar epithelium (Ep) and peripheral connective cells. Along the distal duct, electron lucent subcuticles(Sc) are well distributed. D -F: The secretor y region is composed of single layer of columnar epithelial cells, and the mature secretory product in glandular epithelium appea rs as almos t spherical granules. Nc: endocutilcle layer . Scale bars indicate 100 µ m (D), 50 µ m (E,F) and 20 µ m (A-C), respectively . final electron-dense granules of the secretory product corner (Vollrath, 2007). Even the most spiders produce originated from the distended cisternae are observed near various kinds of silks, which are indifferently used for the the microvilli of the glandular epithelium (Fig. 4F). diverse silken constructs, the agelenid spiders use silk from the ampullate glands for making draglines, frame threads and web building (Kovoor, 1987; Nentwig and Heimer, DISCUSSION 1987). The araneomorph funnel-web spiders of the family Like the other web-building spiders (Peters and Kovoor , Agelenidae include the common grass spiders as well as the 1991; Park and Moon, 2002; Moon and Tillinghast, 2004; venomous European hobo spider (Coddington, 1986). The Moon an d An , 2006), the ampullate gland s were the lar gest web of agelenid funnel-web spider consists of a densely one in this Agelenidae spider . Previous reports have shown packed sheet of silk, which has a long, silken tube in one that there are two pairs of ampullate glands for the genus ANIMAL CELLS AND SYSTEMS Vo l. 12 No. 2 113 Jong-Gu P ark and Myung-Jin Moon Fig. 4. Transmission electron micrographs of the silk producing apparatus in the funnel web spider Agelena limbata . A: Duct has exocuticle, subcuticle (Sc), endocuticle (Ec), and epithelial cells (Ep). B-C: Several types of secretory granules are seen in the cytoplas m of the glandula r epithelial cells. D-E: Broad cisternae of the rough ER (arrow) are found in the epithelium. Small vesicles are synthesized from the rER, and secretory vesicles are formed by fusion with small vesicles F: The apices of the glandular cells are fringed with short and irregular microvilli (Mv). Lu: lumen, Nu: nucleus. All scale bars indicate 5 µm. Araneus (Tillinghast and Townley, 1986; Townley et al.,ampullate glands (major and minor) produce different types 1 9 9 1 ; M o o n an d T i l lin gh ast , 2 0 0 4), g enu s N e p hil a (T il lin gh ast of silk materials (Casem et al., 1999; Blackledge and and Christenson, 1984) and the other Araneidae spiders Hayashi, 2006), the major ampullate gland of A. limbata is (Peters and Kovoor , 1991). Ho we v er , there are fo u r pairs in morphologically very similar to the minor ampullate gland A. limbata similarly to those of wandering Salticidaeexcept for its spigot size and spinneret connection. (Kov oo r , 1 9 8 7), Theri dii da e (Mo on a n d An, 20 06 ), Age len id ae Following the observations of Peakall (1964) on silk (Park and Moon, 2002), and Thomisidae (Moon and An, synthesis in Araneus diadematus, the regulation of 2005) spiders. secretory protein release in the ampullate silk glands has Our previous work has revealed that two pairs of major been frequently considered (Peakall, 1965; Bell and ampullate glands in A. limbata send secretory ductules to Peakall, 1969; Tillinghast and Townley, 1986), since the the anterior spinnerets, and another two pairs of minor property of silk fiber is deeply related with the nature of ampullate glands supply the median spinnerets (Park and secretory silk. Unlike to other araneid spiders (Bell and Moon, 2002). Although it has been known that two kinds of Peakall, 1969; Peters and Kovoor, 1991; Moon and 114 ANIMAL CELLS AND SYSTEMS Vol. 12 No. 2 Ampullate Silk in F unnel-web S pider Tillinghast, 2004), both morphological and anatomical REFERENCES characteristics of the ampullate silk gland in A. limbata are very unique, because its storage sac is spherical instead of Bell AL and Peakall DB (1969) Changes in fine structure during silk protein production in the ampullate gland of the spider normal ampullar appearance. In addition, its secretory tail is Araneus seri cat u s. J C e ll Bi o l 42: 284-295. not in a simple tubular but in a branched tubular appearance. Blackledge TA and Hayashi CY (2006) Silken toolkits: biomechanics Once Kovoor (1987) repoted this kind of spherical of silk fibers spun by the orb web spider Argiope argentata ampullate glands in M ygalomo rphae sp ider . (Fabricius 1775). J Exp Biol 209: 2452-2461. Protein synthesis within the glandular epithelium of the Candelas GC and Cintron J (1981) A spider ( Nephila clavipes ) major ampullate silk glands have been revealed using fibroin and its synthesis. J Exp Zool 21 6 : 1- 6. various experimental techniques including both a cholinergic Candelas GC and Lopez F (1983) Synthesis of fibroin in the c u lt ur ed glan ds o f Nephi la cla v ip es. Comp B ioch e m Ph ysi o l B stimulation (Peakall, 1964, 1965) and mechanically pulling 74: 637-642. stimulation (T illinghast and T ownley , 1986, ’87; Moon and Casem ML, Turner D, and Houchin K (1999) Protein and amino Tillinghast, 2002, 2004). Recently, Moon and Tillinghast acid composition of silks from the cob weaver, Lat r o d ect us (2002) has demonstrated that several kinds of secretory hesperus (black widow). Int J Biol Macromol 24: 103-108. granules are p roduced after mechanical pulling stimulation , Coddington JA (1986) The monophyletic origin of the orb web. and electron densities of the granules were gradually In: Shear WA (ed), Spider Webs and Spider Behavior. increased according to their maturation. In A. limbata, we Stanford Univ Press, pp 319-363. could observe several types of secretory granules at the Coddington JA and Levi HW (1991) Systematics and evolution of spiders (Araneae). Ann Rev Ecol Syst 22: 565-592. secretory region of the ampullate silk gland. Thus, it is very evident that granular modification involves the progressive Gosline JM, Denny MW , and DeMont ME (1984) Spider silk as rubber. Nat u r e 309: 551-552. maturation of the secretory product within the glandular Groome JR, T ownley MA, de T schaschell M, and T illinghast EK epithelium. (1991) Detection and isolation of proctolin-like immuno- It has been also revealed that the secretory silks of the reactivity in Arachnids: Possible cardioregulatory role for ampullate glands in A. limbata are produced by way of proctolin in the orb-weaving spiders Ar gi ope and A r aneu s. J rough endoplasmic reticulum (rER) of each glandular Insect Physiol 37: 9-19. epithelial cell. This extensive rER occupies the whole Kaplan D (1998) Fibrous proteins-silk as a model system. Polym remaining space of the epithelium, however no definitiveDegrad Stab 59: 25-32. Golgi apparatus appeared. Thus, it has been observed that Kovoor J (1987) Comparative structure and histochemistry of silk-producing organs in Arachnids. In: Nentwig W (ed), the Golgi complex does not seem to play an important role Ecophysiology of Spiders. Springer-Verlag, Berlin, pp 159- either concentrating or packaging process of silk production 1 86. in this silk glands, Previously , Bell and Peakall (1969) first Lucas F (1964) Spiders and their silks. Disco very 25: 20-26. reported this kind of unique phenomenon in the ampullate Moon MJ and An JS (2005) Spinneret microstructure of silk gland of the spider Araneus sericatus. Our results also spinning apparatus in the crab spider, Misumenops tricuspidatus suggest that the secretory silk is rapidly produced via rER (Araneae: Thomisidae). Entomol Res 35: 67-74. in a form which is ready for release and undergoes no Moon MJ and An JS (2006) Microstructure of silk apparatus of further step for remodeling via Golgi apparatus (Moon andthe comb-footed spider, Achaearanea tepidariorum ( A r a neae: Theridiidae). Entomol Res 36: 56-63. Tillinghast, 2002). Moon MJ and Tillinghast EK (2002) Fine structure of the Our microstructural analysis using the transmission glandular epithelium during secretory silk production in the electron microscopy (TEM) also suggest that the secretory black widow spider, Latrodectus mactans . Kor J Biol Sci 6: silk is produced by vesicular fusion in a form which is 3 27- 33 3. ready for secretion, and undergoes no further concentration. Moon MJ and Tillinghast EK (2004) Silk production after Candelas and Cintron (1981) and Candelas and Lopez mechan ical pul lin g stimulat io n in t he ampu ll ate sil k gla nds of (1983) also reported that the secretory product of the largethe barn spider, Araneus cavaticus. Entomol Res 34: 123-130. ampullate glands of Nephila clavipes migrates as one Nentwig W and Heimer S (1987) Ecological aspects of spider webs. In: Nentwig W (ed), Ecophysiology of Spiders. homogeneous band in denaturing electrophoresis, and the Springer-Verlag, Berlin, pp 211-225. luminal contents comprise the liquid silk. Obviously, the Park JG and Moon MJ (2002), Fine structural analysis of the silk glandular epithelial cell loses the secretory granules from spinning apparatus in the funnel-web spider , Agelena limbata its cytoplasm during the process of silk release. Thus, it (Araneae: Agelenidae). Kor J Entomol 32: 223-232. seems that the silk proteins produced within epithelial cells Peakall DB (1964) Effects of cholinergic and anticholinergic of the ampullate glands are released by the typical drugs on the synthesis of silk fibroins of spiders. Comp mechanism of merocrine secretion. B i ochem P h y si o l 12: 465-470. ANIMAL CELLS AND SYSTEMS Vo l. 12 No. 2 115 Jong-Gu P ark and Myung-Jin Moon Peakall DB (1965) Regulation of the synthesis of silk fibroins of W (ed), Ecophysiology of Spiders. Springer-Verlag, Berlin, sp id e r s a t th e gla n du la r le v e l. Co mp B io c hem Ph ysiol 15: 509 -pp 203-210. 51 5. Townley MA, Horner NV, Cherim NA, Tugmon CR, and Peters HM and Kovoor J (1991) The silk-producing system of T illinghast EK (1991) Selected aspects of spinning apparatus Linyphia triangularis (Araneae: Linyphiidae) and some development in Araneus cavaticus (Araneae: Araneidae). J comparisons with Araneidae: Structure, histochemistry andMorphol 208: 175-191. function. Z oom orpho log y 111: 1-17 Vollrath F and Selden P (2007) The role of behavior in the Tillinghast EK and Christenson T (1984) Observations on theevolution of spiders, silks, and webs. Annu Rev Ecol Evol Syst chemical composition of the web of Nephila clavipes38: 819-846. (Araneae: Araneidae). J A r ach nol 12 : 69 - 7 4 . Work RW (1981) Web components associated with the major Tillinghast EK and Townley M (1986) The independent ampullate silk fibers of orb-web building spiders. Trans Am regulation of protein synthesis in the major ampullate glands Mi cr osc So c 100: 1-20. of Aran eus cavat i cu s Keyserling. J Insect Physiol 32: 117- W ork R W (1984) Duality in major ampullate silk and precursive 12 3. material from orb-web building spiders (Araneae). Trans Am Tillinghast EK and Townley M (1987) Chemistry, physical Mi cr osc So c 103: 113-121. properties, and synthesis of Araneidae orb webs. In: Nentwig [Received April 30, 2008; accepted May 19, 2008] 116 ANIMAL CELLS AND SYSTEMS Vol. 12 No. 2
Journal
Animal Cells and Systems
– Taylor & Francis
Published: Jan 1, 2008
Keywords: fine structure; silk apparatus; funnel‐web spider; Agelena limbata
