Larval Labial Glands of the Sweet Potato Hornworm, Agrius Convolvuli: Morphology and Secreted Proteins

Journal of Insect Biotechnology and Sericology 78, 53-60 (2009)

Larval labial glands of the sweet potato hornworm, Agrius convolvuli: morphology and secreted proteins

Xinmin Zhaa, Koji Shirai a,＊, Katsuya Yoshikawaa , Shigeru Satob, Rensuke Kanekatsua and Kenji Kiguchia

a Division of Applied Biology, Faculty of Textile Science and Technology, Shinshu University, Ueda, Nagano 386-8567, Japan b Central Institute for Electron Microscope Research, Nippon Medical School, Sendagi, Bunkyo-ku, Tokyo 113-8603, Japan (Received November 13, 2008; Accepted December 8, 2008)

The gross and cell morphology and ultrastructure of larval labial glands of the sweet potato hornworm, Agrius convolvuli, were investigated by light, fluorescent and transmission electron microscopy. The labial gland is a narrow tubular epithelium that can be divided into three regions composed of hexagonal cells, each with a large branched nucleus. The ultrastructure of these cells was similar to that of silk gland cells in silkworms, characterized by well-developed rough endoplasmic reticulum and Golgi bodies. Agrius larvae spin small amounts of fibrous material immediately after hatching and at the wandering stage. Several bands (approx. 30, 67, 70, 116, 200 kDa) were detected by SDS-PAGE from the labial glands throughout the fifth instar stage. Interestingly, most peptides disappeared at around the onset of the wandering stage, replaced by two proteins of approximately 180 and 220 kDa. Two major polypeptides with similar molecular mass (180, 220 kDa) were also detected in fibrous materials spun during the wandering stage. These results suggest that a switchover of protein synthesis occurs in the glands before metamorphosis. Keywords: Agrius convolvuli, larval labial gland, morphology, fibrous material, switchover of protein synthesis

early stage of embryonic organogenesis (Ando and INTRODUCTION Kobayashi, 1996). While these invaginations elongate In 1994, we developed a continuous rearing system of backward and differentiate into silk glands in some in- sweet potato hornworm, Agrius convolvuli, for use as a sects, in most insects they become salivary glands that new experimental insect in comparative studies with other produce saliva (Eichenseer et al., 1999), sometimes con- lepidopterans such as tobacco hornworm, Manduca sexta, taining a variety of volatile components (Svensson and and the silkworm, Bombyx mori (Kiguchi and Shimoda, Bergstrom, 1979). Labial glands of sweet potato horn- 1994). Since then, A. convolvuli has been studied exten- worm, Agrius convolvuli, are the homologous organs with sively in regards to its physiology (Iwami et al., 1996; silk glands of Bombyx mori, and are often referred to as Matsushita et al., 2002; Shimoda et al., 2007), biochemis- labial-silk glands. In the silkworms, labial glands differen- try (Shimoda and Saito, 1997; Lee et al., 1999; Yu et al., tiate into giant exocrine glands named silk glands, which 2002) and neurobiology (Ando et al., 2002; Wang et al., produce a large quantity of silk proteins, mainly in the 2008). last larval instar stage (Suzuki, 1977; Prudhomme et al., The position of hawk moths (Sphingidae) has been 1986; Sehnal and Akai, 1990). Conversely, the glands of classified within the Bombycoidea superfamily (Pittaway, Agrius larvae are narrow and poorly-developed glandular 1993). Although several studies have treated the moths as ducts secreting only small amounts of silk-like material a separate superfamily, most researchers have recognized whose components have not yet been investigated. a close relationship between Sphingidea and Bombycoidea The purpose of this study was to describe the morphol- (Hodges, 1971; Kuznetsov and Stekolnikov, 1985). ogy of labial glands of the sweet potato hornworm, Agri- Despite their close taxonomic affiliation, the survival us convolvuli and its secreted proteins. We found that the strategies of these taxa differ slightly with respect to gross and cell morphology and ultrastructure of labial development: for example, a characteristic feature of silk- glands were essentially similar to those of silk glands in worm development is that they spin cocoons. Bombyx lar- the silkworms. However, we also found a switchover of vae spin silk mainly at the end of the final instar stage to protein synthesis prior to larval-pupal transformation. produce cocoons in which they pupate, while Agrius larvae dig holes in the soil for pupation. MATERIALS AND METHODS The labial glands of most insects arise from a pair of invaginations occurring on the labial segment during the Insects The larvae of Agrius convolvuli used in this study were *To whom correspondence should be addressed. obtained from a laboratory colony established from wild Fax: +81-268-21-5331. Tel: +81-268-21-5334. pupae collected in 1989 at Tsukuba, Ibaraki, Japan Email: [email protected] (K. Shirai) (Kiguchi and Shimoda, 1994). Successive descendants 54 Zha et al. were maintained on artificial diets (Silkmate L4M, Nosan) fifth instar larva. While one pair of forceps was usedto with dried sweet potato leaf powder (10% w/w,1st to 4th lift up the posterior end of the posterior gland, another instar ; 5% w/w, 5th instar) in our laboratory. Larvae were pair was used to squeeze the gland gently from the poste- reared in a group until the end of the fourth instar . After rior to the anterior end until the contents of the lumen that the fifth instar larvae were reared individually ina were extruded onto a sheet of parafilm. For analysis of lu- plastic cup at 25°C under a 12 h light-12 h dark photope- men proteins from the three different gland regions, the riod. labial glands were cut into three regions based on their gross morphology as shown in Fig. 2A, and the proteins Tissue preparation secreted from each region were collected using the same Fifth instar or wandering larvae were anaesthetized by method described above. Samples were taken from the immersion in water before operation. Labial glands were fifth instar day 1 larvae to the wandering stage day 2 lar- removed by dissection through a longitudinal incision on vae daily and stored in −30°C until use. the dorsal integument in 0.75% w/v of NaCl solution. Ad- hering fat body and trachea were carefully removed when Collection and extraction of fibrous material pro- necessary. teins At onset of wandering, larvae were placed on cellulose Optical and Fluorescent microscopy powder with appropriate moisture contained in a plastic To identify the boundary between the anterior and mid- case. After pupation, the fibrous material in the pupal dle region of labial gland, the DNA of the gland cells was chamber was collected into a 1.5 ml tube and extracted as stained by Feulgen staining. Then the boundary was ob- described by Kao and Case (1985). Briefly, the collected served by optical microscopy. material was dissolved in a 0.1 ml extraction buffer con- To observe cell-to-cell boundaries of labial glands, the taining 6 M guanidine hydrochloride, 40 mM Tris-HCl glands of the fifth instar day 2 larvae were removed and buffer (pH 8.8), and 100 mM dithiothreitol for up to 1 h incubated with fluorescent dye (0.5 μg 4’6 -diamidino-2- with occasional vortexing, and then 10 mg of iodoacet- phenylindole (DAPI) /ml PBS) at 37°C for 15 min and amide was added. Incubation at room temperature for 1 h mounted on a glass slide and visualized under a fluores- in the dark was done. The supernatant of extract after cent microscope with green fluorescence filter (Olympus centrifugation for 5 min at 12,000 g was precipitated over- BX 60, Japan). night in four volumes of acetone, and the proteins were harvested by centrifugation at 12,000 g for 5 min. The Observation of fibrous materials in the pupal pellet was resuspended in 30 μl of 10 mM Tris-HCl buffer chamber (pH 7) and stored at −30°C until use. To observe inside of the soil chamber during pupation, a wandering larva was placed into a plastic container Protein determination and SDS-PAGE (15 cm × 15 cm × 30 cm) containing soil. The soil cham- After determining the quantity of protein by Bradford’s ber was formed in a week. Internal surface of the soil method (1976), protein samples were analyzed by SDS- chamber was observed by an Optiphoto FX-21 micro- PAGE as described by Laemmli (1970) under reducing or scope (Nikon, Japan) to determine whether any fibrous non-reducing conditions. Samples were run on 5.0, 7.5 materials had been secreted by the larvae. and 12.5 % gels together with protein molecular mass standards (GE Healthcare Bioscience). Gels were stained Transmission electron microscopy (TEM) using coomassie brilliant blue R-250. For TEM observations, the labial glands of the fifth instar and wandering larvae were fixed in 2.5% glutaralde- RESULTS hyde and post-fixed in 1% osmium tetroxide. The samples were dehydrated in graded series of alcohol and embed- Fibrous material spun by Agrius larvae ded in Epok 812. Ultrathin sections were cut, stained with To understand the function of the labial glands in the uranyl acetate and lead citrate, and examined under a sweet potato hornworm, A. convolvuli, we examined transmission electron microscopy (JEM1010, Nihon whether the larvae spin fibrous materials during their Dennshi). growth and development. It is known that newly hatched larvae spin small amount of fibrous materials (Fig. 1A), Collection of lumen proteins but we have not yet observed any spinning behavior until Lumen proteins for SDS-polyacrylamide gel electropho- the wandering stage. Thereafter, secreted fibrous material resis (SDS-PAGE) were collected as follows. A pair of la- has been observed on the inner surface of the test tubes bial glands were carefully removed to avoid injury to the (1.9 cm) containing wandering larvae (Fig. 1B), as well as Morphology and protein secretion of labial glands in Agrius convoluvuli 55

staining (Fig. 2B). The middle region is straight and located from the forth to the sixth segments, and the posterior region is loosely coiled and located from the seventh to the ninth segments. The posterior region is characterized by its dorso-lateral location and well distributed with a tracheal network. The labial glands develop rapidly during the final fifth instar and reach maximal size at thewan- dering stage (data not shown). DAPI staining of the labial gland in the fifth instar day 2 larvae indicated that each gland consists of a double layer of glandular epithelium (Fig. 2C), which closely resembles the glands of silkworms. Although the gland develops continuously throughout the larval life, the number of cells remains constant. The anterior, middle and posterior regions each consist of approximately 140, 300 and 400 cells, respectively, and each cell possesses a large branched nucleus with a high degree of polyploidy (Fig. 2B, C).

Ultrastructural observation of labial gland cells Ultrastructural observation of labial gland cells from the anterior, middle and posterior regions was performed by TEM in the fifth instar day 2 (Fig. 3) and wandering day 0 larva (Fig. 4). The paired anterior regions are slender ducts that lie in the ventro-thoracic region. The duct cells of the anterior portion are mainly characterized by well-developed microvilli (mi), elongated mitchondria (m) and a thick cuticular intima (ci), as shown in Fig. 3A. The surface of the intima facing the lumen is covered by a thin dense-layer, which resembles the epicuticular layer of the larval cuticle. The cells in the middle and posterior regions of the gland have extremely convoluted nuclei, which contain numerous nucleoli (n), well-developed Golgi bodies (gc), Fig. 1. Fibrous material spun by A. convovuli. A, Fibrous and rough endoplasmic reticulum (rER), in the fifth instar material by a freshly hatched larva. B, Fibrous material at the day 2 larvae (Fig. 3B, C). inner surface of a tube by a wandering larva. Fibrous material is shown by arrows. C, Fibrous material in the pupal chamber In wandering day 0 larva, many Golgi bodies were observed by optical microscopy. observed both in the middle and posterior duct cells (Fig. 4A, C). A number of large vesicles were found in- on the inner surface of pupal chambers after three days side middle duct cells (Fig. 4B) and the number was larg- (Fig. 1C). Thus, it appears that Agrius larvae spin small er than that in the posterior duct cells (Fig. 4D). Fibrous amounts of fibrous material soon after hatching and at the materials were observed in the lumen of both, in the mid- wandering stage. dle and posterior ducts (Fig. 4B, D). The large dense globules (g) in the apical region of the cytoplasm are con- Morphology and cytology of the labial gland sidered to be secreted fibrous globules (Fig. 4D), and they A schematic illustration of a labial gland from a full- are similar to the globules observed in the lumen. grown larva is shown in Fig. 2A. The gland resembles the silk gland of silkworms, Bombyx mori, except that it is Developmental changes in the composition of lu- slender. The paired gland extends from the spinnaret (la- men proteins during the fifth instar bium) to the ninth larval segment. Based on its gross mor- Changes in the protein composition in the lumen of la- phology, it is divided into three regions—anterior, middle bial glands of larvae at different developmental stages and posterior regions. The boundary between the anterior were studied by SDS-PAGE on 7.5% gel (Fig. 5A) and and middle regions is well identified as shown by Feulgen 12.5 % gel (Fig. 5B). Major protein bands (30, 67, 70, 56 Zha et al.

Fig. 2. Gross morphology of larval labial gland of A. convovuli. A, Schematic representation of a half of a pair of labial glands from fifth instar larva. The gland could be divided into three regions, the anterior, middle and posterior region, based on morphological features. B, The junction between anterior and middle region of the labial gland was identified by Feulgen staining. An arrow indicates this junction. C, Morphology of nuclei from posterior region of the labial gland revealed by DAPI staining. Cell boundaries are shown by arrows. Arrowheads indicate branched nucleus.

116, 200 kDa) were detected from days 1-5 of fifth instar. DISCUSSION From onset of the wandering stage these bands suddenly disappeared, and two new dominant polypeptides (180, Morphological characteristics of labial glands 220 kDa) appeared and continued to exist during the wan- The sweet potato hornworm, A. convovuli, is a closely dering stage by SDS-PAGE under both non-reducing con- related species to the tobacco hornworm, M. sexta. The ditions (Fig. 5) and reducing conditions (data not shown). gross morphology of the labial gland from Agrius larva is In addition, the lumen proteins recovered from middle and similar to that of M. sexta (Eaton, 1987). In fact, both posterior regions of the labial glands were collected sepa- taxa resemble in many respects and high homologies of rately and analyzed by SDS-PAGE. No significant differ- several proteins, such as cecropin D (Lee et al., 1999), in- ence was observed in the protein composition between the secticyanin (Saito and Shimoda., 1997) and cytochrome c two regions (data not shown). oxidase subunit 1 (Uno et al., 2004). These members of Fibrous material was recovered from the pupal cham- the sphingidae do not produce cocoons, but bury them- ber, constructed in moist cellulose powder, then the pro- selves in the soil and construct pupal chambers. Agrius tein components of the fibrous materials were extracted. larvae spin a small amount of fibrous material immediate- The solidified fiber was difficult to re-dissolve in aSDS ly after hatching, and similar fibrous materials are found sample buffer. So, guanidine hydrochloride was used for on the inner surface of the pupal chamber. Both of them solubilization of the fiber. The extract was subjected to are secreted from labial glands. SDS-PAGE. The main bands consisted of two proteins of The morphology of the silk gland, which is a homolo- approximately 180 and 220 kDa (Fig. 6), indicating that gous organ to the labial gland, has been well studied in a the components of the fibrous material spun by wandering variety of insect species. Nakagawa (1949) attempted to larvae are almost identical to the proteins recovered from classify the silk glands of various insects, including sever- the lumen of the labial glands. The proteins of fiber in the al sphingid species, based on tracheal shape and its distri- pupal chamber were slightly larger than 180 and 220 kDa bution into 5 types (0, I, II, III and IV). He also classified proteins. The slight modifications to the proteins might the glands into 3 types (N, NZ, and Z type) according to have occurred just before secretion. the shape of the gland. Bombyx silk glands belong to types II and NZ. Sehnal and Akai (1990) described 6 types of the gland shapes in several lepidopteran insects: Morphology and protein secretion of labial glands in Agrius convoluvuli 57

Fig. 3. Ultrastructure of labial gland cells in the fifth instar day 2 larva. A, Cross-section of anterior labial gland cell. B, Middle labial gland cell. C, Posterior labial gland cell. b, basal membrane; gc, Golgi body; n, nucleus; m, mitchondria; mi, microvilli; rER, rough endoplasmic reticulum.

Fig. 4. Ultrastructure of labial gland cells in a wandering stage larva. A, Cross-section of middle labial gland cell near basal membrane. B, Middle labial gland cell near lumen. C, Posterior labial gland cell near the basal membrane. D, Posterior labial gland cell near the lumen. b, basal membrane; g, large dense globule; gc, Golgi body; l, lumen; mi, microvilli; n, nucleus. 58 Zha et al.

Fig. 5. Lumen protein profiles from the fifth instar day 0 larva to wandering stage day 1 larva stained by coomassie brilliant blue R-250. A, 7.5% SDS-PAGE gel. Lanes 1 to 5 contain lumen samples from the fifth inster days 0, 1, 2, 3, and 4 larvae respectively. Lanes 6 and 7 contain lumen samples (36 μg) from wandering stage days 0 and 1 larvae. M1 is high-molecular mass marker. B, 12.5% SDS-PAGE gel. Lanes 1 to 5 contain lumen samples (36 μg) from the fifth instar days 0, 1, 2, 3, and 4 larvae, respectively. Lanes 6 and 7 contain lumen samples from the wandering stage days 0 and 1 larvae. M 2 is low-molecular mass marker.

is generally similar to that of Bombyx silk glands (Akai, 1965; 1984) and other lepidopteran silk glands (Sehnal and Akai, 1990; Sorensen et al., 2006). The apical cell surface with microvilli is separated from the lumen. There is a large branched nucleus with numerous nucleoli in each cell. Signs of proteosynthesis are detected in the cytoplasm of the cells in fifth instar larvae day 2 and wandering stage day 0. Golgi bodies are scattered through out the cytoplasm of cells in fifth instar larvae day 2. These characteristics suggest that the labial gland of Agrius is a primitive type of silk glands. To ensure a safe place for pupation, sphingid insects may have developed the strategy to construct subterranean pupal chambers in the soil instead of silkworm-like cocoons. This development may consequently have stunted the ability to synthe- size silk proteins in this family. Agrius labial glands resemble Bombyx silk glands, not Fig. 6. SDS-PAGE (5% gel) of lumen proteins and compo- only with respect to gross morphology, but also ultrastruc- nents of fibrous material recovered from pupal chambers. ture. The fibrous materials might be encoded by -the ho Lane 1: lumen proteins from the posterior region of labial mologous gene. But differences in gene regulation and gland at wandering stage day 2. Equivalent amounts of protein (1/5 individual worth) were applied to SDS-PAGE. Lane 2: selective splicing may have facilitated the secretion of fi- lumen proteins from the middle region of labial gland at wan- brous materials for disparate environments. dering stage day 2. Equivalent amounts of protein (1/5 individual worth) were applied to SDS-PAGE. Lane 3: fibrous Switchover of synthesis of protein species materials recovered from the pupal chamber. Two bands of 180 and 220 kDa proteins are indicated by arrows. M is high- Biochemical and molecular mechanisms of synthesis molecular mass marker. and secretion of silk proteins have been extensively investigated in the domesticated silkworm, B. mori. The prima- Pieris rapae, Barathra brassicae, Bombyx mori, Anther- ry component of silk proteins, fibroin, is exclusively aea yamamai, Diacrisia subcarnea and Euprotis flava. synthesized in the posterior silk gland and another silk The labial gland of Agrius convovuli clearly belongs to protein, sericin in the middle silk gland. the Bombyx type by tracheal distribution and its shape. In addition to Bombycidae, the core of silk fiber is typ- The Bombyx silk gland is an exocrine organ producing ically composed of heavy chain fibroin (H-fibroin), light considerable quantities of silk proteins. Although Agrius chain fibroin (L-fibroin), and P25 in several other lepi- labial glands are a narrow tublar organ, their ultrastructure dopteran families, including Pyralidae (Zurovec et al., Morphology and protein secretion of labial glands in Agrius convoluvuli 59

1992), Lasiocampidae, and Papilionidae (Tanaka and ACKNOWLEDGMENT Mizuno, 2001). L-fibroin and P25 proteins have not been detected in Saturniid moths Samia cynthia ricini, A. per- This work was supported by the Grant-in-Aid for the nyi and A. yamamai (Tamura and Kubota, 1989; Tanaka Global COE Program (B-019) and the Grant-in Aid for and Mizuno, 2001). In the Trichoptera (Caddisflies), the Young Scientists (B) (12760037 and 14760032) from The core of the silk is primarily composed of H-fibroin and L- Ministry of Education, Culture, Sports, Science and Tech- fibroin (Yonemura and Sehnal., 2006). However, in anoth- nology (MEXT), Japan. The authors are indebted to the er non-mulberry silkworm, Samia cynthia ricini, fibroin Division of Gene Research of the Research Center for proteins are comprised of two polypeptides (97 and 45 kDa) Human and Environmental Sciences, Shinshu University that are linked by a disulfide bond and these are immuno- for the utilization of facilities. chemically distinct from Bombyx mori (Ahmad et al., 2004). REFERENCES Similarly, while we assumed that fibroin and sericin would be detected in very small quantities in the silk from Akai, H. (1965) Studies on the ultrastructure of the silkworm, Agrius, no protein components with molecular weights Bombyx mori L.. Bull. Seric. Exp. Sta., 19, 375-484 (in Jap- matching the fibroin and sericin of B. mori were observed anese with English summary). Akai, H. (1984) The ultrastructure and function of the silk in the fifth instar larva or the wandering stages larva. The gland cells of Bombyx mori. 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