_??_1991 by Cytologia , Tokyo Cytologia 56: 679-685 , 1991

Ultrastructure of Gland of Webspinners, Oligotoma japonica (Insecta, Embioptera)

Takayuki Nagashima, Nao Niwa, Shuji Okajima and Toshifumi Nonaka

Laboratory of Entomology, Tokyo University of Agriculture, Sakuragaoka 1-1-1, Setagaya, Tokyo 156, Japan AcceptedSeptember 12, 1991

Certain Chelicerata and in the Arthropoda produce silk threads of a aceous material which is insoluble in water . All glands producing silk are considered of ec todermal origin. Silk glands of chelicerates are primary epidermal invaginations, which often occur in several pairs differing in their kind of secretion, and the silk glands of most (labial glands) are derived secondarily from other types of glands. However, a characteristic feature of the Embioptera including lower Hexapoda is the retention of the silk glands in the tarsal segment of the front legs; these glands are considered derivatives from the hypodermic and are covered with bristles (Sefnal and Akai 1990). The silk webbing is employed in build ing silken tunnels which they inhabit. So far as the authors are aware, there are few papers concerning the structure of the spinning organ of the Embioptera (Melander 1903, Mukerji 1927, Alberti and Storch 1976, Nagashima et al. in press). As part of an ongoing study, the ultrastructure of the silk gland of the Japanese webspin ner, Oligotoma japonica Okajima is described here in detail. Also touched on is the silk protein synthesis and intracellular transport.

Materials and methods

The material used in this study consisted of adult males of Oligotoma japonica okajima captured in Ishigaki Island of Okinawa Prefecture and Miyazaki city of Miyazaki Prefecture in Japan. For transmission electron microscope (TEM), the enlarged 1st tarsal segments, after having been carefully cut out of the forelegs of the insects, were immersed for 4hr at room temperature in a prefixative solution of 2% paraformaldehyde and 2.5% glutalaldehyde. The tarsal segments were then thoroughly rinsed with 0.1M cacodylate buffer and postfixed for

2.5hr at room temperature in cacodylate-buffered (pH 7.5) 1% OsO4 solution. After de hydration, the tarsal segments were embedded in Epon 812. Golden tissue sections, cut with a glass knife on the Reichert OMU2 ultramicrotome and double stained with uranyl acetate and lead citrate, respectively, were observed with a JEM 100-CX electron microscope. Semi thin sections (0.75ƒÊm thick) were mounted on a glass slide and stained with Azur B prior to observation under a standeard light microscope. For scanning electron microscope (SEM), the tarsal segments were dehydrated in an ethanol/t-butyl alcohol series, and dried by freeze dryer. Specimens were examined with a Hitachi S-2400 SEM. The number of glands was counted after cold 20% KOH treatment overnight.

Results and discussion

General features On the ventral surface of the enlarged Ist (Fig. 1) and the small 2nd tarsal segments of the 680 TakayukiNagashima et al. Cytologia56

forelegs are a number of hollow cuticular processes like a spine with no socket which are presumably modified microtrichia; these are found only the ventral surface of these segments. Each of the cuticular processes (e. g., Ejaktor of Barth 1954, and Spinnborste of Alberti and Storch 1976) is connected with the glandular chamber by a fine duct (Melander 1903, Mukerji 1927, Barth 1954, Alberti and Storch 1976). Own results, however, revealed no communica tions between their duct and the cuticular process. In Oligotoma japonica over 80 individual glands are present in the enlarged 1st segment, and these are aligned in approximately three files in the longitudinal section. The ducts of the 1st segment which are situated close to the 2nd segment extend to the ventral cuticule of

Fig. 1. Scanning electron micrograph of the whole fore tarci, showing the enlarged 1st segment

(tar 1). tar 2; 2nd segment of tarsus, tb; tibia. Scale: 200ƒÊm. Fig. 2. Light micrograph of the 1st (tar 1) and 2nd (tar 2) tarsal segments in longitudinal section. The silk glands(*) are situated only in the 1st segment. Scale: 100ƒÊm.

the I st segment because the 2nd segment holds no silk gland (Figs. 2, 3). The epidermal cells of the ventral side are present in higher density in than those on the dorsal side (Figs. 2, 3). Each glandular chamber (extracellular cavity or lumen) is globular in form, with a dia meter of approx. 60ƒÊm, and bounded by a single layer (approx. 20ƒÊm) of gland cells .

Glandular chamber The glandular chamber or lumen forms a voluminous reservoir of viscid secretions, and accumlates numerous silk (fibroin and sericin) (Fig. 6). These proteins are clearly separated into two layers in the lumen: the outer layer (approx.

2-4ƒÊm) is occupied sericin protein and the inner layer contains a large quantity of fibroin pro- 1991 Ultrastuructureof SilkGland of Webspinners 681 tein (Fig. 6). The thickness of a sericin layer is not uniform , and in some areas the layer is even unrecognizable. Orientation of the fibroin fibers in the lumen is random and no re markable mass of fibers was observed in this work (Fig . 6).

Fig. 3. Schematic structure of the 1st (tar 1) and 2nd (tar 2) tarsal segments, showing an ar rangement of silk glands (sg) and ducts (du). cp, cuticular process; ec, epidermis cell; ecc, extra cellular cavity; sgn, nucleus of silk gland.

Fig. 4. Transverse section of a silk gland at middle level showing multinuclear cell and embranc ing a voluminous extracellular cevity (ecc). G, Golgi complex; mc, mitochondria; sgn, nucleus of silk gland. Scale: 3ƒÊm. 682 TakayukiNagashima et al. Cytologia56

Gland cell The glandular cell of Oligotoma japonica is one large (Figs. 3, 4), multinuclear cell com posed of a voluminous extracellular cavity or gland lumen as found in Haploembia solieri and Embia (=Monotylota) ramburi (Alberti and Storch 1976), and 8 to 12 nuclei are usually observed in each cell. It is well known that caterpillars of and of certain sat urnids produce commercial silk, and that the main components of this silk are fibroin and sericin. These components have separate secretory functions: the elementary fibroin fibers are secreted (synthesized) in the posterior region and the sericins are synthesized in the posterior division of the middle silk gland (Akai 1976). Although, the components of silk protein in Oligotoma japonica are also presumably fibroin and sericin, the characteristic feature of the silk gland of this is that both of them are produced from a single cell.

Fig. 5. Part of the gland cell showing well developed rough endoplasmic reticulum (rer) and Golgi complexes (G) containing fibroin fibers (arrows). ecc, extracellular cavity; mc, mitochondria;

mv, microvilli. Scale: 2ƒÊm.

Fig. 6. Inner part of the gland cell and the lumen, showing the sericin (sf) and fibroin (ff) fibers . mv, microvilli; rer, rough endoplasmic reticulum. Scale: 1ƒÊm.

Characteristic ultrastructures of intracellular organelles of the tarsal silk gland are granular endoplasmic reticulum (ER) and Golgi complex which are thought to be involved in the fibroin and sericin synthesis. Well developed granular ER is observed throughout the gland cell (Figs. 4, 5). The Golgi vacuole contains elementary fibroin fibers (Figs. 4, 5) as observed in the silk worm (Akai 1976) and orbweb (Plazaola and Candelas 1991), and the free (isolated) fibroin globules are moved inside (the gland lumen side). The discharge into the gland lumen of the fibroin fibers is achieved by exocytosis of a gland cell. Numerous secretory granules, presumably sericin material are widely distributed inside 1991 Ultrasturucture of Silk Gland of Webs pinners 683

Fig. 7. Longitudinal section of the base of duct, showing the ducteole cell (dc). du, ducteole; ecc,

extracellular cavity; ip, inner process; sg, silk gland cell. Scale: 2ƒÊm. Fig. 8. Higher magnification of the base of duct, showing inner processes (ip) constructed by a

docteole cell. ecc, extracellular cavity; mc, mitochondria; sg, silk gland cell. Scale: 1ƒÊm.

Fig. 9. Longitudinal section of the duct, showing cuticular tinning composed of several cuticle layers. Inset: enlargement. ff, fibroin fibers; sf, sericin fibers. Scale: 1ƒÊm (0.2ƒÊm in inset). 684 TakayukiNagashima et al. Cytologia56 the gland cells, however, the sericin globules and Golgi vacuoles containing elementary sericin strings as observed in Bombyx mori (Akai 1976) are not seen in silk gland cells of this insect. The inner surface of the gland contains many microvilli of which structure of contents is similar to that of accumulation in glandular chamber. This seems to suggest that the sericin is not secreted by exocytosis as occurs in the silkworm but is transported along microvilli to the gland lumen. According to Plazaola and Candelas (1991), structural changes of the microvilli are seen in the silk gland of the orbweb spider, Nephia clavipes the luminal membrane of a stim ulated gland (at peaking metabolically activation) appears totally depleted of microvilli of electron dense appearance.

Fig. 10. Diagramatic representation of glandulas unit of Oligotoina japonica. ecc, extracellular cavity; du, ducteole; dc, ducteole cell er, endoplasmic reticulum; g, Golgi complex; me, mito chondria; sg, silk gland; sgn, nucleus of silk gland.

Duct Each glandular chamber has a fine duct (ducteole) which is connected to the hollow cut icular process like spine on the ventral surface of the 1st and 2nd tarsal segments. The ducteole is a single tube and has not branched off in the tarsal segments.

In addition to a gland cell, at the base of duct is a single cell which supports the base of along duct. The supporting cell surrounds the base of the ducteole, and is connected with a gland cell (Figs. 7, 10). This cell has a complicated inner process (Figs. 7, 8), presumably a hand (fingers) or a petal-like structure which is composed of cuticule-like materials. The cytoplasmic components of the cell are characterized by the absence of a complex Golgi ap paratus such as that found in the silk gland cell (Fig. 7). The epithelial lining of the ducteole observed in other gland is lacking; however, in its place cuticular lining (thickness is about 0.43ƒÊm) composed of several (about 7) cuticule layers (Fig. 9).