Morphology and Ultrastructure of the Salivary Glands of the Spittlebug Lepyronia coleopterata (L.) (Hemiptera: Aphrophoridae) Author(s): Haiying Zhong, Yalin Zhang and Cong Wei Source: Zoological Science, 31(4):213-222. 2014. Published By: Zoological Society of Japan DOI: http://dx.doi.org/10.2108/zs130215 URL: http://www.bioone.org/doi/full/10.2108/zs130215

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ZOOLOGICAL SCIENCE 31: 213–222 (2014)

¤ 2014 Zoological Society of Japan

Morphology and Ultrastructure of the Salivary Glands of the Spittlebug Lepyronia coleopterata (L.) (Hemiptera: Aphrophoridae) Haiying Zhong, Yalin Zhang, and Cong Wei * Key Laboratory of Plant Protection Resources and Pest Management, Ministry of Education, Entomological Museum, Northwest A&F University, Yangling, Shaanxi 712100, China

We investigated the salivary glands in Lepyronia coleopterata (L.), and found that the salivary glands are paired structures and consist of principal and accessory glands. Each principal gland contains an anterior lobe and a posterior lobe. Three types of acini (I, II, III) are observed in the anterior lobe, whereas the posterior lobe contains only one type of acini (IV). Rhabdus emerges from the middle portion of the acini III and IV. The oval-shaped accessory gland connects with the principal gland via a long duct. The long duct consists of a slightly coiled basal segment and a highly convoluted distal segment, with the terminal end of the latter constricted and connected with the accessory gland. A slightly convoluted transparent tube connects with the accessory gland at the former’s distal end. The accessory gland, accessory salivary duct and the accessory salivary tube are observed for the first time in spittlebugs. Ultrastructurally, each type of acinus is made up of one type of secretory cells, but the rhabdus comprises two types of cells. Secretory granules in different type of cells are different in size, shape and electron density, which indicate either different materials are synthesized or these materials undergo a process of maturation. The rhabdus is empty in structure and contains several channels, with the lumen filled with abundant fine granular materials. Fine dark granules existed in the periphery of some secretory granules are probably virus particles. Microorganisms are observed in the cells of the acini I, III and rhabdus. Key words:

principal gland, accessory gland, ultrastructure, morphology, Cercopoidea

INTRODUCTION The superfamily Cercopoidea (Hemiptera: Cicadomorpha) comprises approximately 3000 described species in 340 genera worldwide (Cryan and Svenson, 2010). This group is well known to include spittlebugs, froghoppers, or cuckoospit insects. The former name is due to the habit of the nymphal spittlebugs immerse themselves in a foamy salivalike mass, a mixture of bubbles trapped in liquid expelled from the alimentary canal and secretions secreted from the Malpighian tubules (Rakitov, 2002). This frothy mass functions to protect nymphs against drought and enemies during their nymphal stages. Although this saliva-like mass does not cause harm to host plants, direct plant damage and/or virus transmission are caused during feeding. For instance, a leaf scorch disease, caused by the bacterium Xylella fastidiosa, is typically vectored by spittlebugs (Almeida and Purcell, 2003; Groves et al., 2005; Sisterson et al., 2008; Janse and Obradovic, 2010; Sanderlin and Melanson, 2010; Zhang et al., 2011). During feeding, spittlebugs inject toxic saliva to plants and degrade the molecular structure of the tissue (Guagliumi, 1972; Gallo et al., 1978; Nakano, 1981). Moreover, toxic enzymes in saliva cause the blockage of conducting chan* Corresponding author. Tel. : +86-29-87092509; E-mail: [email protected] doi:10.2108/zs130215

nels and intoxication (Nunes and Camargo-Mathias, 2006). The saliva production is closely related to the salivary glands. Thus the pathogen transmission by spittlebugs during feeding makes it very necessary to understand the structure and physiology of the salivary glands of these species. To date, there are very few reports on the salivary glands of spittlebugs. Pioneering descriptions of the gross morphology of the salivary glands of one spittlebug species in West Indies, Tomaspis saccharina, were made by Kershaw (1914). His paper was concerned mainly with the digestive system (including salivary glands) of T. saccharina, which possesses 12 tubular glands and a greatly long, coiled, chitinous salivary reservoir with a small irregularly-shaped flattened gland lying in the distal end. Later, Cecil (1930) studied the salivary glands of the spittlebug Philaenus leucophthalmus at the morphological and histological levels, which demonstrated the salivary glands are two pairs parallel to the oesophagus. Recently, Nunes and Camargo-Mathias (2001) and Roma et al. (2003) investigated the salivary glands of the spittlebugs Mahanarva fimbriolata and M. posticata, respectively. More recently, Nunes and CamargoMathias (2006) investigated the ultrastructure of the salivary glands of the M. fimbriolata in detail. These reports have provided a valuable guide for further studies of the salivary glands of spittlebugs from the standpoint of the ultrastructure. However, a reasonable question whether the accessory gland of spittlebugs joins the principal gland via a thin accessory salivary duct as other

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hemipterans (Baptist, 1941; David 1977; Tsai and Perrier, 1993, 1996; Wayadande et al., 1997; Serrão et al., 2008; Zhang et al., 2012; Zhong et al., 2013a, b) remains unclear. In the present study, we investigate the salivary glands of male Lepyronia coleopterata, a widely distributed species in China. We are aiming to provide a detailed morphological and ultrastructural description of the salivary glands for this species, which will be informative to further comparative, taxonomic, phylogenetic and functional investigations for spittlebugs. MATERIALS AND METHODS Spittlebug Lepyronia coleopterata were collected from the grass weeds around a pond (type locality, 108°7′E, 34°30′N, elev. 520 m), Yangling, Shaanxi Province, China in June 2010. The individuals were anesthetized by chilling before beginning a dissection. Light microscopy The salivary glands were dissected out in phosphate buffered saline (PBS, 0.2 M, pH 7.2) under a Stereoscopic Zoom Microscope Motic SMZ168. Photographs were taken by a scientific digital micrography system equipped with an Auto-Montage Imaging system and a Qimaging Retiga 2000R digital camera (CCD).

Fig. 1. Gross morphology of the salivary glands of the spittlebug Lepyronia coleopterata. pg, principal gland; ag, accessory gland; I– IV, four types of acini of the principal gland; r, rhabdus; psd, principal salivary duct; asd, accessory salivary duct; ast, accessory salivary tube.

Scanning electron microscopy Principal salivary ducts of the salivary glands and saliva syringe were fixed in 2.5% glutaraldehyde (PBS, 0.1 M, pH 7.2). After four rinses (15 min each time) with 0.1 M phosphate buffered saline (pH 7.2), the materials were dehydrated in an ethanol series, dried in a critical point-drier and coated with gold. Then the samples were examined and photographed under a JEOL JSM-6360 LV SEM operated at 15 kV. Transmission electron microscopy The salivary glands were fixed in 2.5% glutaraldehyde (PBS, 0.1 M, pH 7.2) for 12 h. The samples were rinsed four times (15 min each time) in 0.1 M PBS, and post-fixed in 1% osmium tetroxide for 1.5 h. After the same rinse, the materials were dehydrated through a graded ethanol series, infiltrated with a mixture of Epon 812 in acetone and were embedded in pure Epon 812. Ultrathin sections were stained with uranyl acetate and lead citrate, and examined under a JEOL JEM-1230 transmission electron microscopy at 80 kV. The terminology of the salivary glands mainly follows that of Tsai and Perrier (1996).

RESULTS Gross morphology of the salivary glands (Figs. 1, 2) The salivary glands of Lepyronia coleopterata are paired structures, which are situated within the head and thorax, and consist of principal and accessory glands. The principal gland (pg) contains an anterior lobe and a posterior lobe. Arrangement of the secreting units of the lobes is shown in Figs. 1 and 2. Three types of acini (I, II and III) are observed in the anterior lobe, whereas the posterior lobe contains only one type of acinus (IV). Nine or twelve blindly ending rhabduses arise from the junction of the acini III and IV. Each rhabdus, approximately 4.0 mm in length, is basally thin, thickening gradually towards the distal portion. There are 7–8 I-type acini, twelve II-type acini, and sixteen to twenty III-type acini. Each type of acini differs from others in shape and size. The I-type acinus is oval, which connects closely, forming a large oval structure. The II- and III-type

Fig. 2. Schematic illustration of the Lepyronia coleopterata salivary glands. Anterior lobe of the principal gland contains three types of acini (I, II, III), whereas the posterior lobe of the principal gland contains only one type of acinus (IV). Rhabdus (r) inserts between the acini III and IV. The highly convoluted segment is pulled straight in order to show its route. An oval-shaped accessory gland (ag) connects at its base with the accessory salivary duct (asd) and distally with the accessory salivary tube (ast). psd, principal salivary duct.

acini arrange concentrically and form a pumpkin-shaped structure, respectively. The IV-type acinus is rounded and contains two large acini and three small acini. Two principal salivary ducts (psd) separately open into the salivary syringe. In freshly dissected individuals, all the acini and the rhabduses are whitish. The accessory gland (ag) is oval-shaped and transparent, which joins the principal gland via a very long accessory salivary duct (asd) inserting into the principal salivary duct between the anterior lobe and the posterior lobe of the principal gland. The accessory salivary duct is long and yellowish brown, containing two distinct regions: a slightly coiled

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basal segment and a highly convoluted distal segment. The slightly coiled segment runs haphazardly between the head and the prothorax. It follows a winding course and makes a number of loops, forming a highly convoluted segment which constricts to become an extremely thin zone, with its distal end connected with the accessory gland when approaches the middle portion of the head capsule. A collapsible translucent tube connects with the accessory gland basally, with distal portion lying freely in the hemolymph of the head. The tube is much shorter but thicker than the accessory salivary duct.

Fig. 3. TEM micrographs of secretory cells that form acini I. (A) Cytoplasm is packed with secretory granules (sg). l, lumen; m, muscles; n, nuclei with evident nucleoli. (B) High magnification of a secretory cell, showing numerous secretory granules (sg) of different electron density, size and shape. (C) Detail of the cytoplasm, showing lamellar rough endoplasmic reticulum (lrer) and secretory granules (sg) of varying size and shape. Microorganisms (as arrow indicated) exist in the lumen (l) of the intracellular canaliculi. (D) Magnification of extensive lamellar rough endoplasmic reticulum (lrer), secretory granules (sg) and microorganisms (as arrows indicated). Scale bars: (A) 5.0 μm; (B, D) 1.0 μm; (C) 2.0 μm.

Ultrastructure of the salivary glands Principal gland Four cellular types of the acini and two types of cells of the rhabdus in L. coleopterata are identified based on their cytoplasmic characteristics. All the cells are externally enveloped by muscle cells. In the I-acinus, cells are occupied by abundant electron-dense secretory granules (Fig. 3A). These secretory granules are different in shape and size, with some granules exhibiting varying densities from the middle area to the periphery (Fig. 3B–D). It appears that smaller secretory granules fuse to become larger ones (Fig. 3B, C). Extensive lamellar rough endoplasmic reticulum (Fig. 3C, D) and large intracellular canaliculi lining with sparse microvilli facing the lumen (Fig. 3A, C) are observed around the secretory granules. Many microorganisms, about 0.2–0.5 μm in diameter and 1.5–2.0 μm in length, are also found either in the cytoplasm or in the lumen of the intracellular canaliculi (Fig. 3C, D). Cells of the II-acinus are characterized by abundant electron-lucent secretory granules and shallow basal infoldings (Fig. 4A). The spherical secretory granules vary in size, with some granules displaying different electron density in the center and the periphery (Fig. 4A,

Fig. 4. TEM micrographs of secretory cells that form acini II. (A) Secretory cell with abundant secretory granules (sg) and elongate nuclei (n). l, lumen of intracellular canaliculi; mv, microvilli; if, infoldings of basal plasma membrane. (B) High magnification of vesicle-liked structures (white arrows) in the lumen of intracellular canaliculi. The structures contain many fine granular materials (asterisks). Black arrow indicates larger granular materials formed by the fusion of some smaller fine granular materials. mv, microvilli. (C) High magnification of secretory granules (sg) and nuclei, which possess large nucleoli (n) and clumps of heterochromatin (h). (D) Magnification of extensive lamellar rough endoplasmic reticulum (lrer) in the cytoplasm. Scale bars: (A) 1.0 μm; (B) 0.5 μm; (C, D) 0.2 μm.

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C). Intracellular canaliculi lined by a layer of short microvilli facing the lumen exist among the secretory granules (Fig. 4A). In the lumen of the intracellular canaliculi, vesicle-like

structures with numerous fine granular materials are observed (Fig. 4B). It seems that smaller vesicle-like structures and fine granular materials fuse to become larger

Fig. 5. TEM micrographs of secretory cells that form acini III. (A) Peripheral portion of a secretory cell with well-developed lamellar rough endoplasmic reticulum (lrer). Muscles (m) externally envelope the secretory cells. (B) The cytoplasm is packed with electronlucent secretory granules (sg1). Large shoeshaped secretory granules (sg2) are electrondense in the middle and electron-lucent at the periphery. n, nuclei. (C) Detail of the cytoplasm, showing lamellar rough endoplasmic reticulum (lrer) and different types of secretory granules (sg1, sg2, and sg3). (D) High magnification of a secretory granule with fine dark granules (as indicated by arrow) in the periphery. (E) Cytoplasm of the secretory cell contains lamellar rough endoplasmic reticulum (lrer), elongated nuclei (n), secretory granules (sg), and microorganisms (arrows). (F) Magnification of the secretory cell showing extensive lamellar rough endoplasmic reticulum (lrer) and ferritin-like granules (f). Scale bars: (A, D, F) 0.5 μm; (B) 5.0 μm; (C, E) 2.0 μm.

Fig. 6. TEM micrographs of secretory cells that form acini IV. (A) Peripheral portion of a secretory cell, showing numerous secretory granules (sg). The intracellular canaliculi bears long microvilli (mv) facing the lumen (l). m, muscles. (B) Middle portion of a secretory cell, showing secretory granules (sg) of different size and shape. Lamellar rough endoplasmic reticulum (lrer) is found around the secretory granules. n, nuclei with evident oval nucleoli; (C) Middle portion of a secretory cell, showing numerous secretory granules (sg). Clusters of fine electrondense granules (as arrow indicated) seem to release white substances into secretory granules. (D) Intracellular canaliculi of a secretory cell bear long dense microvilli (mv). Irregular shaped secretory granules (sg) aggregate adjacent to the intracellular canaliculi. Scale bars: (A) 2.0 μm; (B, C, D) 1.0 μm.

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Fig. 7. TEM micrographs of secretory cells that form rhabdus. (A) Cross-section through the rhabdus. The secretory cells contain numerous secretory granules (sg) and large area of lamellar rough endoplasmic reticulum (lrer). These secretory granules enter into the lumen via the apex of the microvilli (mv). The lumen of the rhabdus is filled with fine granular materials (asterisks). m, muscles. (B) Detail of the first type of cells, showing secretory granules (sg) and lamellar rough endoplasmic reticulum (lrer). The secretory granules enter into the lumen via the apex of the microvilli (mv). White arrows indicate intracellular microorganisms, and black arrows show fine granular materials in the rhabdus lumen. (C) High magnification of microorganisms existing in the first type of cells. (D) Detail of the second type of cells, showing numerous microorganisms (as arrows indicated) and large area of vesicular rough endoplasmic reticulum (vrer). (E) Cytoplasm of the second type of cells, showing large amount of vesicular rough endoplasmic reticulum (vrer), secretory granules (sg) of different electron density. Scale bars: (A, B) 2.0 μm; (C) 0.5 μm; (D, E) 1.0 μm.

Fig. 8. Micrographs of the principal salivary duct and accessory gland. (A, B) Scanning electron microscopy (SEM) micrographs of the principal salivary duct. Note the outer membrane (om) covers the principal salivary duct (psd) facing the hemolymph. (C) Crosssection of the principal salivary duct. The basal plasma membrane invaginates into deep infoldings (if). Peripheral area of a secretory cell contains numerous vesicles (v). bl, basal lamina (D) Apical region of a cell of the principal salivary duct. Secretory products (as arrow indicated) are visible in the lumen (l). (E) Cross-section of the ovalshaped accessory gland. The basal plasma membrane invaginates into well-developed infoldings (if). sg, secretory granules. (F) Cytoplasm of a secretory cell of the ovalshaped accessory gland, showing extensive lamellar rough endoplasmic reticulum (lrer), clusters of vesicles (v) and oval nuclei (n). Scale bars: (A, B) 10.0 μm; (C, D) 1.0 μm; (E) 0.2 μm; (F) 2.0 μm.

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ones, respectively. Elongate nuclei with large nucleoli and scatter of secretory granules with different electron density clumps of heterochromatin are also visible at the basal (Fig. 7D, E). Large number of double-membraned microorregion of the cells (Fig. 4A, C). Extensive lamellar rough ganisms, about 3.5 μm in diameter and 1.0–2.0 μm in endoplasmic reticulum exists around the secretory granules length, are observed near both two types of rough endoplas(Fig. 4D). mic reticula (Fig. 7B–D). Cells of the III-acinus are characterized by extensive lamellar rough endoplasmic reticulum and numerous secrePrincipal salivary duct tory granules. The lamellar rough endoplasmic reticulum The principal salivary duct is covered by an outer memeven reaches to the shallow invaginations of the basal brane (Fig. 8A, B), which throws into deeply penetrating plasma membrane (Fig. 5A). Most secretory granules are infoldings (Fig. 8C). Electron-lucent vesicles of different size electron-lucent, oval and vary in size (Fig. 5B, C). Some and shape are scattered throughout the cytoplasm (Fig. 8C). secretory granules are electron-lucent center and electronIt appears that the vesicles release secretory substances dense periphery, with fine dark granules (about 56.0 nm in into the lumen of the duct (Fig. 8D). diameter) in the periphery (Fig. 5C, D). Large shoe-shaped secretory granules (about 10.0 μm in diameter and 22.0 μm Accessory gland and accessory salivary duct in length) with electron-dense center and electron-lucent The accessory gland joins the principal gland via an periphery are scattered sparsely and randomly in the cytoaccessory salivary duct. The duct is divided into two differplasm (Fig. 5B). Clusters of rod-shaped microorganisms are ent regions based on their morphological and cellular feafound near the elongated nuclei containing small clumps of chromatin (Fig. 5E). Ferritinlike granules are visible adjacent to the large area of lamellar rough endoplasmic reticulum (Fig. 5E, F). Cells of the IV-acinus are also featured by lamellar rough endoplasmic reticulum and abundant secretory granules (Fig. 6A, B). However, distinct from the cells of the acini II and III, all of the secretory granules in the acinus IV are highly electron-dense and greatly vary in size and shape (Fig. 6A–C). Some smaller secretory granules appear to fuse into larger ones (Fig. 6B, C). Adjacent to the secretory granules, nuclei with large nucleoli and small clumps of chromatin are also observed (Fig. 6B). Clusters of fine electron-dense granules appear to release white substances into a large secretory granule (Fig. 6C). Many deform secretory granules aggregate in the vicinity to intracellular canaliculi that are tightly packed with long microvilli facing the lumen. It appears that secretory materials are released by these granules into the lumen (Fig. 6A, D). The rhabdus is a hollow structure, with several channels filled by fine granular materials (Fig. 7A, B). The channel wall of the rhabdus comprises two types of cells, with their apical border coated by short sparse microvilli (Fig. 7A, B). Cells of the first type contain Fig. 9. TEM micrographs of the oval-shaped accessory gland and accessory salmany electron-dense secretory granules and ivary duct. (A) Extensive lamellar rough endoplasmic reticulum (lrer) is seen in the extensive lamellar rough endoplasmic reticucells of the accessory gland. (B) Apical part of the highly convoluted segment cells of the accessory gland duct. The microvilli (mv) are long and dense. Numerous lum (Fig. 7A, B). The secretory granules are vesicles (v) and mitochondria (mi) are obvious in the cytoplasm. Asterisks indicate different in size, shape and electron density, secretion in the lumen (l). (C) Cross-section of the slightly coiled segment of the with some granules are electron-dense in the accessory gland duct, showing many intracellular canaliculi lined by dense center and electron-lucent at the periphery microvilli (mv). n, nuclei; if, infoldings of basal plasma membrane; mi, mitochon(Fig. 7A, B). Fine granular substances can be dria; l, lumen. (D) Magnification of the slightly coiled segment cells of the accesobserved to be released into the channel by sory gland duct, showing well-developed basal infoldings (if) and abundant secretory granules distributed among the mitochondria (mi). Filiform materials (as arrow indicated) exist in the duct lumen microvilli (Fig. 7B). Being different from the first and intracellular canaliculi lumen. v, vesicles; mv, microvilli. (E) Magnification of a cellular type, notable features of the second cell of the slightly coiled segment of the accessory gland duct, showing lamellar rough endoplasmic reticulum (lrer). mi, mitochondria; mv, microvilli; n, nuclei. type of cells are the rich development of the Scale bars: (A, B, E) 0.5 μm; (C, D) 2.0 μm. vesicular rough endoplasmic reticulum and the

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tures: a highly convoluted segment and a slightly coiled segment (for details, see below). Basal area of the accessory gland cells appears to be compartmentalized by well-developed basal infoldings, with a few electron-dense secretory granules scattered nearby (Fig. 8E). Extensive rough endoplasmic reticulum, some electron-lucent vesicles and oval-shaped nuclei with clumps of chromatin are visible in the cytoplasm (Figs. 8F, 9A). Cells of the highly convoluted segment possess well-developed infoldings at the basal region and bear dense long microvilli at the apical border (Fig. 9B). Many mitochondria and electron-lucent vesicles exist throughout the cytoplasm (Fig. 9B). Cells of the slightly coiled segment have welldeveloped apical microvilli and basal infoldings associated with mitochondria (Fig. 9C, D). Many intracellular canaliculi coated by dense microvilli facing the lumen are obvious in the cytoplasm (Fig. 9C, D). Filiform materials are observed in the lumens of the coiled segment and intracellular canaliculi (Fig. 9D). The cytoplasm contains abundant mitochondria, nuclei, a few vesicles, and lamellar rough endoplasmic reticulum (Fig. 9C–E). DISCUSSION The morphology of the salivary glands in Cicadomorpha To date, the salivary glands in spittlebugs have been investigated only in limited species, i.e., Tomaspis saccharina (Cercopidae) (Kershaw, 1914), Philaenus leucophthalmus (Aphrophoridae) (Cecil, 1930), Mahanarva fimbriolata (Cercopidae) (Nunes and Camargo-Mathias, 2001, 2006) and Mahanarva posticata (Cercopidae) (Roma et al., 2003). We add one species, Lepyronia coleopterata, for its gross morphology and fine structure. We conclude that the common characters shared by the four cercopoid species (i.e., T. saccharina, M. fimbriolata, M. posticata, L. coleopterata) are as follows: the principal gland consists of an anterior lobe and a posterior lobe, with the former being composed of three types of acinous secreting units (acini I, II, III), and the latter possessing only one type of secreting units (acini IV); the rhabduses (previously termed “filamentous portion”) insert between the acini III and IV. However, the salivary glands of P. leucophthalmus remarkably differ from those of the abovementioned spittlebugs in general structure, which consist of a cylindrical upper pair and a club-shaped lower pair, and the duct of each gland runs anteriorly and fuses within the head. In addition, no other portions, e.g., the accessory gland and rhabduses, are observed in the salivary glands of P. leucophthalmus. Such a great difference probably attributes to the related species belonging to different genera, or to the careless observation of the author. The morphology of the salivary glands in spittlebugs resembles that described for some other species in Cicadomorpha (Kershaw, 1914; David, 1977; Tsai and Perrier, 1996; Wayadande et al., 1997; Nunes and CamargoMathias, 2001; Roma et al., 2003; Zhang et al., 2012; Zhong et al., 2013a, b). However, morphological differences of the principal and accessory glands within Cicadomorpha also exist. In Cicadoidea, the principal gland contains many digitate secreting units; the accessory gland comprises a convoluted accessory salivary tube, a gular gland, and an accessory salivary duct (David, 1977; Zhong et al., 2013a, b). In Membracoidea, such as the leafhoppers Dalbulus

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maidis, Graminella nigrifrons, and Psammotettix striatus, the secreting units constituting the principal gland are acinous, and the accessory gland is elbow-, boot-, oblong-, oval- or rod-shaped (Tsai and Perrier, 1996; Wayadande et al., 1997; Zhang et al., 2012). These variations of the salivary glands within Cicadomorpha might provide useful information for further taxonomic and phylogenetic analysis of related groups. Is the rhabdus really an accessory gland? Nunes and Camargo-Mathias (2001) and Roma et al. (2003) respectively termed the rhabdus (previously termed “filamentous portion”) of the salivary glands in M. fimbriolata and M. posticata as accessory gland, which is more than two in number. However, it is noted that the salivary glands of many hemipterans possess a pair of accessory glands with each joining at the junction of the anterior and posterior lobes of the principal gland via a duct (i.e., accessory salivary duct) (Baptist, 1941; David, 1977; Tsai and Perrier, 1996; Wayadande et al., 1997; Nunes and CamargoMathias, 2001; Roma et al., 2003; Zhang et al., 2012; Zhong et al., 2013a, b). Furthermore, the accessory salivary duct is lined with a layer of cuticle facing the lumen (David, 1977; Zhong et al., 2013a, b). In our investigation, apart from rhabduses observed in the salivary glands of L. coleopterata, other elements, i.e., a long accessory salivary duct, an ovalshaped accessory gland and a collapsible accessory salivary tube, are also found, and the junction of the duct and the tube is indicated by the rod-shaped accessory gland. The layout of the accessory glands of L. coleopterata is similar to that of other species in Hemiptera. At the ultrastructural level, cells of the rhabdus of L. coleopterata are occupied by two types of extensive rough endoplasmic reticula and abundant electron-dense secretory granules, and the basal region of the cells is devoid of infoldings. These characters indicate large amounts of enzyme products are synthesized. In addition, the absence of basal infoldings and the existence of very sparse apical microvilli coated the channel wall suggest that the products are more viscous. Herein, we conclude that the rhabdus is not the accessory gland based on its features. Function of the salivary glands in spittlebugs Most hemipterans, feeding on xylem or phloem sap, secrete lipo-proteinaceous gelling saliva, which forms a supportive and protective salivary sheath surrounding the stylets as they inject into the plant tissues (Crews et al., 1998; Miles, 1972; Backus et al., 2005). They also secrete watery saliva consisting of different types of enzymes (Miles, 1972). Our results suggest that the principal gland and accessory gland of L. coleopterata produce at least two types of secretions and possess probably different roles according to the cytoplasm appearance and the content of the gland lumen: the saliva secreted from the principal gland is viscous and plays a role in the formation of salivary sheath; the saliva secreted from the accessory gland is more watery and plays a part in feeding. The principal glands of L. coleopterata have secretory cells rich in organelles engaged typically in high metabolic activity and synthesis of proteinaceous materials, i.e. extensive rough endoplasmic reticulum and numerous secretory

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granules, as has been indicated by Palade (1975). This morphological character suggests that the principal glands are the site where enzymatic products of the saliva are manufactured, as has been suggested for the primary salivary glands of many species in Hemiptera (Baptist, 1941; Miles, 1960, 1972; Ghanim, 2001; Reis et al., 2003; Swart and Felgenhauer, 2003; Swart et al., 2006; Azevedo et al., 2007). Secretory granules in the same secretory cells of L. coleopterata exhibit differences in shape, size and electron density, suggesting that different material may be synthesized. The presence of different secretory cells indicates materials synthesized experience a maturation process, as has been indicated for the salivary glands of the planthopper Peregrinus maids (Ammar, 1986) and for M. fimbriolata (Nunes and Camargo-Mathias, 2006). Apart from the above mentioned features, cells of the II-acinus and III-acinus of the principal gland in L. coleopterata also possess shallow basal infoldings, which can modify the gland content by increasing the surface for ion exchange and water transport from the hemolymph via the cell (Serrão and Cruz-Landim, 1996, 2000). Serrão et al. (2008) suggested the translucent appearance of the principal gland content is caused by the large quantities of water in the saliva due to the presence of basal infoldings, which leads the water transport from the hemolymph to the gland lumen. Two types of peculiar secretory granules are observed in the acini III of L. coleopterata for the first time in our study, and their nature and function need to be investigated further. In contrast, many characters found in the accessory gland of L. coleopterata are related to cells engaged in water transport, i.e., well-developed basal infoldings, many electron-lucent vesicles, and a few electron-dense secretory granules. These cytoplasmic features indicate that the accessory gland likely either functions in osmoregulation or plays an important role in diluting saliva, as has been indicated for the salivary glands of many hemipterans (Baptist, 1941; Miles, 1960, 1972). The existence of extensive rough endoplasmic reticulum in the accessory glands cells suggests enzymic secretions are synthesized here. Our results suggest that many fine dark granules are present in the periphery of some secretory granules of the acini-III secretory cells. These fine particles are morphologically similar to those observed in the salivary glands of the planthopper Peregrinus maidis, aphids Rhopalosiphum padi and Metopolophium dirhodum, glassy-winged sharpshooter Homalodisca vitripennis and whitefly Bemisia tabaci (Ammar, 1987; Peiffer et al., 1997; Cicero and Brown, 2008; Stenger et al., 2009), and also in the alimentary canal of the leafhopper Cicadulina mbila, brown planthopper Nilaparvata lugens, and aphid Rhopalosiphum padi (Noda et al., 1991; Gray and Banerjee, 1999; Ammar et al., 2009). Ghanim et al. (2001) speculated that the principal gland of whitefly Bemisia tabaci is responsible for manufacturing, storing, and discharging various enzymic products; the accessory gland is the ideal environment to “absorb virions from the hemolymph, compartmentalize them in vesicles, and deliver them with the watery saliva”, since the gland absorbs and transports materials produced “elsewhere in the insect’s body”. We speculate that the fine dark particles observed in the electron-dense periphery of secretory granules of the acini-III secretory cells in the present

study are virus particles, which can be absorbed from the hemolymph, compartmentalized in the secretory granules, and inoculated into plants hosts when L. coleopterata salivates during feeding. Cells of the slightly coiled segment of the accessory salivary duct in L. coleopterata are densely packed with microvilli at their apical border, and the basal region of the cells has well-developed infoldings associated with mitochondria. Similar observations were reported in the salivary glands of the thrip Heliothrips haemorrhoidalis by Del Bene et al. (1999), who proposed that these cells are specialized in the manufacture of a fluid secretion. This would, therefore, imply the duct of L. coleopterata is involved in the further dilution of the proteinaceous secretion synthesized by the rough endoplasmic reticulum. In comparison, cells of the highly convoluted segment of the accessory salivary duct in L. coleopterata have abundant electron-lucent vesicles, well-developed apical microvilli and basal infoldings. The cell organization and features of the two duct regions in L. coleopterata may suggest that the concentration and content of the secretion in these zones are different, and the secretion in the highly convoluted segment of the accessory salivary duct is more watery. This speculation is further supported by the finding of two different luminal contents in corresponding duct regions of L. coleopterata. The different luminal content probably attributes to the dilution of the secretion (Serrão et al., 2008). Therefore, we reason that the watery saliva produced in the accessory gland is diluted in the highly convoluted segment before being discharged out, and then followed by the subsequent elaboration in the slightly convoluted segment. The existence of many mitochondria in the cells of the two duct regions indicates a high activity in water movement engaged in saliva dilution, as has been indicated by Serrão et al. (2008) for the salivary glands of the bed bug Cimex hemipterus. Different insect organs (such as Malpighian tubules and other organs in the reproductive and digestive systems) have been reported to harbor microorganisms (Sacchi et al., 2008; Crotti et al., 2010; Zhong et al., 2013a), which play different role in insects survival, such as nutrient provision, influence on development, reproduction and speciation, natural enemy defense and immunity (Dale and Moran, 2006). Kwaik (1996) suggested that the vesicular rough endoplasmic reticulum might provide sufficient nutrients for microorganisms. In our study, microorganisms are observed in the acini I, III and rhabdus of the salivary glands of L. coleopterata. This probably suggests these regions are suitable for microorganisms’ survival, since they are susceptible to food quality, structural and physiological factors (Crotti et al., 2009, 2010). However, the identity and function of these microorganisms need to be studied further to clarify the interactions between L. coleopterata and related microorganisms. ACKNOWLEDGMENTS The authors thank Mr. Qinglong Li (Northwest A&F University, Yangling, China) for assistance in collecting insect specimens. We sincerely thank Prof. John Richard Schrock (Emporia State University, USA) and anonymous reviewer(s) for revising this manuscript and providing valuable comments. This research is supported by Program for New Century Excellent Talents in Universities (NCET10-0691) and the Key Project of Introducing Top Overseas Cultural

Salivary glands of a spittlebug & Educational Experts to the Northwest A&F University Founded by the Chinese Ministry of Education (Grant No. TS2011XBNL061).

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