- Email: [email protected]
Anatomy and fine structure of the alimentary canal of the spittlebug Lepyronia coleopterata (L.) (Hemiptera: Cercopoidea)
Arthropod Structure & Development 42 (2013) 521e530
Contents lists available at SciVerse ScienceDirect
Arthropod Structure & Development journal homepage: www.elsevier.com/locate/asd
Anatomy and fine structure of the alimentary canal of the spittlebug Lepyronia coleopterata (L.) (Hemiptera: Cercopoidea) Haiying Zhong, Yalin Zhang*, Cong Wei* Key Laboratory of Plant Protection Resources and Pest Management, Ministry of Education, Entomological Museum, Northwest A&F University, Yangling, Shaanxi 712100, China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 15 March 2013 Accepted 30 April 2013
The alimentary canal of the spittlebug Lepyronia coleopterata (L.) differentiates into esophagus, filter chamber, midgut (conical segment, tubular midgut), and hindgut (ileum, rectum). The filter chamber is composed of the anterior extremity of the midgut, posterior extremity of the midgut, proximal Malpighian tubules, and proximal ileum; it is externally enveloped by a thin cellular sheath and thick muscle layers. The sac-like anterior extremity of the midgut is coiled around by the posterior extremity of the midgut and proximal Malpighian tubules. The tubular midgut is subdivided into an anterior tubular midgut, midmidgut, posterior tubular midgut, and distal tubular midgut. Four Malpighian tubules run alongside the ileum, and each terminates in a rod closely attached to the rectum. Ultrastructurally, the esophagus is lined with a cuticle and enveloped by circular muscles; its cytoplasm contains virus-like fine granules of high electron-density. The anterior extremity of the midgut consists of two cellular types: (1) thin epithelia with well-developed and regularly arranged microvilli, and (2) large cuboidal cells with short and sparse microvilli. Cells of the posterior extremity of the midgut have regularly arranged microvilli and shallow basal infoldings devoid of mitochondria. Cells of the proximal Malpighian tubule possess concentric granules of different electron-density. The internal proximal ileum lined with a cuticle facing the lumen and contains secretory vesicles in its cytoplasm. Dense and long microvilli at the apical border of the conical segment cells are coated with abundant electron-dense fine granules. Cells of the anterior tubular midgut contain spherical secretory granules, oval secretory vesicles of different size, and autophagic vacuoles. Ferritin-like granules exist in the mid-midgut cells. The posterior tubular midgut consists of two cellular types: 1) cells with shallow and bulb-shaped basal infoldings containing numerous mitochondria, homocentric secretory granules, and fine electron-dense granules, and 2) cells with well-developed basal infoldings and regularly-arranged apical microvilli containing vesicles filled with fine granular materials. Cells of the distal tubular midgut are similar to those of the conical segment, but lack electron-dense fine granules coating the microvilli apex. Filamentous materials coat the microvilli of the conical segment, anterior and posterior extremities of the midgut, which are possibly the perimicrovillar membrane closely related to the nutrient absorption. The lumen of the hindgut is lined with a cuticle, beneath which are cells with poorly-developed infoldings possessing numerous mitochondria. Single-membraned or doublemembraned microorganisms exist in the anterior and posterior extremities of the midgut, proximal Malpighian tubule and ileum; these are probably symbiotic. Ó 2013 Elsevier Ltd. All rights reserved.
Keywords: Alimentary tract Morphology Ultrastructure Auchenorrhyncha Aphrophoridae
1. Introduction As the second most species-rich superfamily in the Cicadomorpha, Cercopoidea includes approximately 3000 described species classified into five families: Cercopidae, Aphrophoridae,
* Corresponding authors. Tel./fax: þ86 29 87092509. E-mail addresses: [email protected] (H. Zhong), [email protected] (Y. Zhang), [email protected] (C. Wei). 1467-8039/$ e see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.asd.2013.04.005
Clastopteridae, Machaerotidae and Epipygidae (Cryan and Svenson, 2010). They are commonly called spittlebugs or froghoppers, and the former name is attributed to the nymphs immersing their soft bodies in a frothy saliva-like mass that protects them from desiccation and predators. Spittlebugs feed exclusively on xylem sap, which is an imbalanced diet and has a low concentration of nutrients (Cheung and Marshall, 1973b; Dickson, 1979). In order to eliminate excessive water from the ingested juice and concentrate the nutrients effectively, part of their alimentary canal modifies into a junction
522
H. Zhong et al. / Arthropod Structure & Development 42 (2013) 521e530
known as “filter chamber”, which consists of the anterior and posterior extremities of the midgut as well as the proximal Malpighian tubules (Marshall and Cheung, 1974). The filter chamber is a shunting complex that can concentrate ingested xylem fluid approximately 10-fold (Cheung and Marshall, 1973b) and phloem sap about 2.5-fold (Lindsay and Marshall, 1981); excessive water can be directly transferred from the anterior extremity of the midgut to the hindgut (Marshall and Cheung, 1974). In addition, the alimentary canal may contain endosymbionts which make a significant contribution to their hosts’ nutrient absorption (Moran et al., 2005). The alimentary canal of spittlebugs has been morphologically and/or histologically investigated for only a limited number of species (Kershaw, 1914; Cecil, 1930; Cheung and Marshall, 1973a; Marshall and Cheung, 1973, 1974). Although these studies have provided important data about the fine structure and cytochemistry of the alimentary canal in those species, more detailed information on the alimentary canal of other species is still needed, and the location of microorganisms that reside either in the lumen or in the cytoplasm of the alimentary canal also merits further investigation. Herein, we investigate the gross morphology and fine structure of the alimentary canal of Lepyronia coleopterata (Linnaeus), a widely distributed species in China, using both light microscopy and transmission electron microscopy. We are aiming to elucidate the alimentary canal at the morphological and ultrastructural levels, and to provide more information for future investigation about the microorganisms that reside in the cytoplasm.
a slender and elongate esophagus (Oe) that runs posteriorly and connects with the filter chamber (FC). The filter chamber is a complex junction with four, clearly defined regions: anterior extremity of the midgut, posterior extremity of the midgut, proximal Malpighian tubules, and proximal ileum. The greatly expanded anterior extremity of the midgut is coiled around by the slender posterior extremity of the midgut and proximal Malpighian tubules. A very thin cellular sheath and thick muscle layers externally enclose the filter chamber. The midgut outside the filter chamber is differentiated into two distinct regions: a considerably inflated saclike conical segment (CS), and a very slender tubular midgut. The tubular midgut has been divided into four regions based on their morphological and ultrastructural characters: a thin anterior tubular midgut (AMg), a very thick mid-midgut (MMg), a thick posterior tubular midgut (PMg), and a thin distal tubular midgut (DMg). It exhibits a “U-shaped” path as follows: the anterior tubular midgut connecting with the conical segment descends backwards and connects with the mid-midgut, which runs back to the posterior tubular midgut; the posterior tubular midgut ascends and connects to the distal tubular midgut entering into the filter chamber. The hindgut emerges halfway along the filter chamber and consists of a long and uniform ileum (Il) and a slightly inflated rectum (Rc). Four Malpighian tubules (MT) run alongside the ileum, and each of them terminates in a rod closely attached to the rectum (Fig. 1).
2. Materials and methods
3.2.1. Esophagus The esophagus is externally enveloped by well-developed circular muscles and internally is lined with a layer of convoluted cuticle (about 0.1 mm) (Fig. 2A). Beneath the cuticle are epithelia resting on a basal lamina (about 2.0 mm thick) (Fig. 2A). The basal plasma membrane of the epithelium invaginates into poorly developed infoldings. In the cytoplasm, mitochondria, elongated nuclei and fine virus-like granules (about 0.07 mm in diameter) of high electron-density are observed (Fig. 2B).
Specimens of male L. coleopterata were collected in Yangling, Shaanxi Province, China, in June 2010 and 2012. 2.1. Light microscopy The live L. coleopterata were anesthetized by chilling and their alimentary canals were dissected out in phosphate buffer (PBS, 0.2 M, pH 7.2) under a Motic SMZ168 Stereoscopic Zoom Microscope. Photographs were taken with a scientific digital micrography system equipped with an Auto-montage imaging system and a Qimaging Retiga 2000R digital camera (CCD).
3.2. Ultrastructure of the alimentary canal
3.2.2. Filter chamber Ultrastructurally, different regions of the filter chamber exhibit significant differences, and their typical characteristics are described in detail below.
2.2. Transmission electron microscopy The dissected alimentary canals were pre-fixed in 2.5% glutaraldehyde (phosphate buffer, 0.2 M, pH 7.2) at 4 C for 12 h, washed five times with 0.1 M phosphate buffer (pH 7.2) and post-fixed in 1% osmium tetroxide for 2 h at 4 C. Then the materials were rinsed five times in the same phosphate buffer (0.1 M, pH 7.2). After dehydration in an ethanol series and 100% acetone and infiltration with a mixture of Epon 812 in acetone (1: 3 (v/v) for 2 h, 1: 1 (v/v) for 5 h, 3: 1 (v/v) for 12 h, separately), the samples were embedded in pure Epon 812 and incubated at 30 C for 24 h, at 60 C for 48 h, respectively. Ultra-thin sections were stained with uranyl acetate and lead citrate, and examined under a JEOL JEM-1230 TEM at 80 kV and photographed. The terminology of the alimentary canal structures follows that of Cheung and Marshall (1973a) and Marshall and Cheung (1974). 3. Results 3.1. Gross morphology of the alimentary canal The alimentary canal of L. coleopterata consists of esophagus, filter chamber, midgut, and hindgut. The alimentary tract begins as
3.2.3. Cellular sheath The thin cellular sheath comprising numerous long and thin cells forms a recticulum-shaped structure. The sheath cells are externally surrounded by well-developed circular muscles with large nuclei and rod nucleoli (Fig. 2C). 3.2.4. Anterior extremity of midgut (the filter chamber proper) The anterior extremity of the midgut consists of two types of cells: thin epithelium, and large cuboidal cells. Both types of cells lie on a thin basal membrane and display great differences at the ultrastructural level. The thin epithelium has the following characters: 1) welldeveloped and regularly-arranged apical microvilli are coated by filamentous materials; 2) basal plasma membrane invaginates into regularly spaced and tubular infoldings; and 3) the cytoplasm is packed with abundant mitochondria (Fig. 2D). The large cuboidal cells feature very short and sparse microvilli at the apical border, and secretory vesicles, secretory granules, mitochondria and rough endoplasmic reticulum exist in the cytoplasm (Fig. 3A). These secretory granules differ in size, shape and electron-density, with some electron-lucent granules containing radial inclusions of high electron-density (Fig. 3A); some secretory
H. Zhong et al. / Arthropod Structure & Development 42 (2013) 521e530
523
Fig. 1. General structure of alimentary canal of Lepyronia coleopterata (L.), with one Malpighian tubule left and the other three tubules removed. AMg, anterior tubular midgut; CS, conical segment; DMg, distal tubular midgut; FC, filter chamber; Il, ileum; MMg, mid-midgut; MT, Malpighian tubule; Oe, esophagus; PMg, posterior tubular midgut; Rc, rectum.
granules are electron-dense in the center and electron-lucent at the periphery (Fig. 3B). Septate desmosomes joining the large cuboidal cells are also visible in the cytoplasm (Figs. 3A, B). A cluster of intracellular single-membraned microorganisms (about 0.25e 0.40 mm in diameter) is observed adjacent to the rough endoplasmic reticulum (Fig. 3C). 3.2.5. Posterior extremity of the midgut Regularly-arranged apical microvilli coated by filamentous materials and wide basal infoldings devoid of mitochondria are characteristics of cells in this gut region. Scattered elements of rough endoplasmic reticulum are observed in the cytoplasm, where lysosome-like structures and double-membraned microorganisms are also contained (Fig. 3D). 3.2.6. Proximal Malpighian tubule (internal Malpighian tubule) The proximal Malpighian tubule consists of a single layer of cells with well-developed and regularly-arranged basal infoldings and apical microvilli (Figs. 2C, 3E and 4A). Double-membraned microorganisms and scattered rough endoplasmic reticulum are observed in the cytoplasm (Fig. 3E). At the apical region of the cell, concentric granules (about 2.0 mm in diameter) are prominent with electron-dense gradations from the center to periphery (Fig. 4A). Some concentric granules display filamentous-like areas in their peripheries (Fig. 4B). Large nuclei with obvious nucleoli exist in the cytoplasm (Fig. 4C). 3.2.7. Internal proximal ileum The internal proximal ileum is lined with a layer of cuticle facing the lumen. Beneath the cuticle are cells containing secretory
vesicles and large nuclei. Tracheoles penetrate the cells and even reach to the cytoplasm (Fig. 4D). 3.2.8. Conical segment Cells of the conical segment rest on a basal lamina, outside of which are well-developed circular muscles (Fig. 5A). Their basal plasma membrane invaginates into well-developed infoldings extending deeply into rough endoplasmic reticulum (Fig. 5B). Mitochondria of different shape are visible around the scattered elements of rough endoplasmic reticulum (Fig. 5B). The apical border of the cell bears long and dense microvilli coated by filamentous materials and aggregations of electron-dense fine granules (Fig. 5C). 3.2.9. Anterior tubular midgut (AMg) The anterior tubular midgut is externally surrounded by conspicuous longitudinal muscles (Fig. 5D). Its cells lie on a thin basal lamina, and are characterized by well-developed basal infoldings associated with mitochondria and dense, regularly-arranged apical microvilli (Fig. 5D). Spherical secretory granules and oval secretory vesicles of different sizes are observed at the basal area of the cells. Mitochondria, sparse rough endoplasmic reticulum, large autophagic vacuoles and small vacuoles exist in the cytoplasm (Figs. 5D and 6A). 3.2.10. Mid-midgut (MMg) Cells of the mid-midgut also lie on a thin basal lamina. At the basal region, their basal plasma membrane invaginates into welldeveloped infoldings associated with mitochondria (Fig. 6B). Rough endoplasmic reticulum is scattered through the cytoplasm,
524
H. Zhong et al. / Arthropod Structure & Development 42 (2013) 521e530
Fig. 2. (A) Transverse section of esophagus lined with a convoluted cuticle (c) facing the lumen (l), thick basal lamina (bl) and basal infoldings (if) are also visible. (B) Detail of the cytoplasm of the esophagus, with aggregations of virus-like fine granules (white asterisks), a thick cuticle (c) and elongate nuclei (N). Secretions (black asterisks) exist in the lumen. (C) Cells of cellular sheath (cs), which are externally surrounded by well-developed circular muscles (mu) containing nuclei (N) with evident nucleoli (n). Basal infoldings (if) and apical microvilli (mv) of the cells of the proximal Malpighian tubule (imt) (arrow) are also obvious. (D) Transverse section of the anterior extremity of the midgut. Filamentous materials (fm) coat the apical microvilli (mv) facing the lumen (l). Basal infoldings (if) are tubular-shaped and regularly-spaced. Abundant mitochondria (mi) exist throughout the cytoplasm. bl, basal lamina. Scale bars: 5.0 mm in (A); 0.5 mm in (B); 2.0 mm in (C, D).
among which are numerous ferritin-like granules, mitochondria, and nuclei (Fig. 6C). The apical microvilli are less dense than those of the anterior tubular midgut (Figs. 5D and 6A, C). 3.2.11. Posterior tubular midgut (PMg) Cells of the posterior tubular midgut are significantly different from cells of other gut regions, and are categorized into two distinct types. The first type of cells is characterized by very shallow and bulb-shaped basal infoldings, and their cytoplasm is packed with secretory granules and mitochondria (Fig. 6D). These secretory granules vary in size and shape, and smaller granules appear to fuse to become larger ones (Figs. 6D and 7A). Scattered elements of rough endoplasmic reticulum and polyploid nuclei with evident
nucleoli are also visible among the secretory granules (Fig. 7A). In the cytoplasm, aggregations of fine electron-dense granules and mitochondria with cristae are observed (Fig. 7B). Compared with the first type of cells, the second type of cells has the following features: their basal infoldings are well-developed and associated with mitochondria; apical microvilli are dense and regularly arranged; large nuclei with evident nucleoli are surrounded by numerous mitochondria, sparse rough endoplasmic reticulum, and secretory vesicles filled with fine granular materials (Fig. 7C, D). 3.2.12. Distal tubular midgut (DMg) Cells of the distal tubular midgut are similar to those of the conical segment, except they lack filamentous materials and
H. Zhong et al. / Arthropod Structure & Development 42 (2013) 521e530
525
Fig. 3. (A) The apical region of the large cuboidal cells of the anterior extremity of the midgut (filter chamber proper). Rough endoplasmic reticulum (rer), septate desmosomes (sd), mitochondria (mi), sparse microvilli (mv), secretory vesicles (sv), and electron-dense secretory granules (sg) are visible. (B) Cytoplasm of the large cuboidal cells of the anterior extremity of midgut (filter chamber proper) contains extensive rough endoplasmic reticulum (rer) and secretory granules (sg) of electron-dense center and electron-lucent periphery. (C) Large cuboidal cells of the anterior extremity midgut (filter chamber proper), showing extensive rough endoplasmic reticulum (rer), electron-dense secretory granules (sg) and microorganisms (arrows). (D) Transverse section of the posterior extremity of the midgut. Regularly-arranged microvilli (mv) are evident. Filamentous materials (fm) cover the apex of apical microvilli. Basal plasma membrane invaginates into wide infoldings (if). The cytoplasm contains microorganisms (arrow), lysosome-like structures (ly) and rough endoplasmic reticulum (rer). (E) Transverse section of the proximal Malpighian tubule, showing well-developed and regularly-arranged basal infoldings (if) and apical microvilli (mv). Scattered rough endoplasmic reticulum (rer) and microorganisms (arrows) are also seen in the cytoplasm. bl, basal lamina; l, lumen; Scale bars: 0.5 mm in (A, B, C); 0.2 mm in (D, E).
electron-dense fine granules covering the apex of microvilli (Fig. 7E). 3.2.13. Hindgut The hindgut contains two parts (ileum and rectum) and is lined with a cuticle facing the lumen. Cells underlying the cuticle possess very poorly developed basal infoldings, while abundant mitochondria exist within the cytoplasm. However, some ultrastructural differences also exist between the ileum and rectum. At the basal area of the ileum cells, double-membraned microorganisms and electron-lucent secretory vesicles of different sizes are observed, and the smaller secretory vesicles seem to fuse to become larger
ones (Fig. 8A). The microorganisms are morphologically similar to those found in the cytoplasm of the posterior extremity of the midgut and the proximal Malpighian tubules, but differ greatly from those detected in the cells of the anterior extremity of the midgut where single-membraned microorganisms reside (Fig. 3C). In the cells of the rectum, however, no elements such as secretory vesicles and microorganisms are observed (Fig. 8B). 4. Discussion The general structure of the alimentary canal of L. coleopterata (L.) observed in this investigation closely resembles that depicted
526
H. Zhong et al. / Arthropod Structure & Development 42 (2013) 521e530
Fig. 4. (A) Transverse section of the proximal Malpighian tubule, showing concentric granules (cg) with different electron-density from the center to the periphery. (B). Apical area of the cells of the proximal Malpighian tubule. Concentric granules (cg) with filamentous periphery are shown. (C) Cells of the proximal Malpighian tubule contain large nuclei (N) with evident nucleoli (n). The apical microvilli (mv) are dense and long. (D) The internal proximal ileum is lined with a layer of cuticle (c) facing the lumen (l), and its cytoplasm contains large nuclei (N) and secretory vesicles (sv). Tracheoles (tr) are well-developed and penetrate into the plasma membrane. bl, basal lamina; if, infoldings of the basal plasma membrane; mi, mitochondria. Scale bars: 1.0 mm in (A, C); 0.5 mm in (B); 2.0 mm in (D).
in previous studies of other spittlebug, leafhopper and cicada species (Licent, 1912; Kershaw, 1914; Cecil, 1930; Cheung and Marshall, 1973a; Marshall and Cheung, 1973, 1974; Zhong et al., 2011). The canal is differentiated into at least four clearly defined regions: an elongate esophagus, a modified filter chamber, a midgut (including a sac-like conical segment and a tubular midgut), and a hindgut. Recently, the perimicrovillar membrane, a lipoprotein membrane, was detected in the midgut cells of some hemipterans, e.g., the spittlebug Mahanarva posticata, the cicada Quesada gigas and the bug Eurygaster integriceps, which coats the microvilli from their base to apex and extends into the gut lumen (Silva et al., 1995, 1996, 2004; Terra, 1988; Fonseca et al., 2010; Mehrabadi and Bandani, 2011). The perimicrovillar membrane exhibits a
function similar to the peritrophic matrix of the midgut as in some other insects with chewing mouthparts (Terra, 1988). Our fine structure investigation found that filamentous materials coat the apical microvilli of cells of the anterior extremity of the midgut, the posterior extremity of the midgut, as well as the conical segment of L. coleopterata (Figs. 2D, 3D and 5C). Similar materials, previously termed “filamentous coats”, have been described as covering the surface or apex of the apical microvilli of the filter chamber proper and midgut of some other species in Cicadomorpha (Cheung and Marshall, 1973a; Marshall and Cheung, 1974; Lindsay and Marshall, 1980). Marshall and Cheung (1974) deduced that such coatings probably function to bind cations (especially potassium) contained in the xylem sap. Filamentous materials observed in our study are possibly the
H. Zhong et al. / Arthropod Structure & Development 42 (2013) 521e530
527
Fig. 5. (A) Transverse section of the conical segment, showing well-developed circular muscles (mu) that envelope the cells, which have well-developed basal infoldings (if) and dense microvilli (mv). Nuclei (N) and abundant mitochondria (mi) are observed in the cytoplasm. (B) High magnification of the basal area of the conical segment cells. Basal plasma membrane invaginates into wide infoldings (if) extending to scattered rough endoplasmic reticulum (rer). Mitochondria (mi) of different shapes exist among the rough endoplasmic reticulum. (C) High magnification of the apical region of the conical segment cells. Filamentous materials (fm) and abundant electron-dense fine granules (asterisks) cover the apex of apical microvilli (mv). (D) Transverse section of the anterior tubular midgut. Cells with well-developed basal infoldings (if) contain abundant mitochondria (mi). Secretory vesicles (sv) and secretory granules (sg) are also evident in the cytoplasm. Microvilli (mv) are dense and regularly-arranged. Nuclei (N) and rough endoplasmic reticulum (rer) scatter in the cytoplasm. l, lumen; mu, muscles; bl, basal lamina; sd, septate desmosomes. Scale bars: 2.0 mm in (A, D); 0.5 mm in (B); 1.0 mm in (C).
perimicrovillar membrane, similar to their morphology; such a membrane should be closely related to nutrient absorption, as in other fluid-feeding hemipterans. Special fine granules of high electron-density are observed in the conical segment cells of the alimentary canal of L. coleopterata (Fig. 5C), which have never been reported for hemipterans, to date. The existence of these granules at the apex of the microvilli of the conical segment suggests that those granules are associated with food digestion and nutrient absorption, since the conical segment has been reported to be one of the regions for nutrient absorption (Cheung and Marshall, 1973a). In addition, secretory granules and/or vesicles are also found in cells of different gut
regions of L. coleopterata, such as the anterior extremity of the midgut, internal proximal ileum, anterior tubular midgut, posterior tubular midgut and ileum (Figs. 3AeC, 4D, 5D, 6D, 7A, C, 8A), indicating that these regions play a vital role in secretion. However, the nature and function of these fine granules needs to be investigated further. Basal infoldings of cells in the anterior extremity of the midgut (filter chamber proper) of L. coleopterata are tubular-shaped and regularly-spaced (Fig. 2D). They are structurally similar to depictions for the cicada Gaeana maculata by Marshall and Cheung (1974), and were speculated to possess an intrinsic rigidity to prevent them from collapsing during contraction of the filter
528
H. Zhong et al. / Arthropod Structure & Development 42 (2013) 521e530
Fig. 6. Electron micrographs of the tubular midgut. (A) Detail of the apical region of the cells of anterior tubular midgut, showing autophagic vacuoles (av) and small vacuoles (v). The cytoplasm contains abundant mitochondria (mi) and scattered elements of rough endoplasmic reticulum (rer). Long apical microvilli (mv) are regularlyarranged and dense. (B) Basal region of mid-midgut cells, showing well-developed basal infoldings (if) associated with mitochondria (mi). (C) Apical parts of the cells of the mid-midgut. The cytoplasm contains ferritin-like granules (f), mitochondria (mi) and sparse rough endoplasmic reticula (rer). The apical microvilli are long but sparse, and some of them are ruptured. (D) The first cellular type of the posterior tubular midgut. Shallow and bulb-shaped basal infoldings (if) are visible. Cytoplasm filled with secretory granules (sg) and mitochondria (mi). The secretory granules are electron-lucent in center and electron-dense at periphery. bl, basal lamina; N, nuclei. Scale bars: 1.0 mm in (A, B, C); 2.0 mm in (D).
chamber (Marshall and Cheung, 1974). However, basal infoldings found in the cells of other gut regions of L. coleopterata are morphologically different from the above-mentioned infoldings, viz., they are not tubular shaped and irregularly-spaced. In addition, basal infoldings found in the gut cells other than in the anterior extremity of midgut (filter chamber proper) are associated with mitochondria. This probably indicates that these cells perform a strong secretory function, as has been suggested for pylorus cells in the leafhopper Eurymela distincta (Lindsay and Marshall, 1980). The hindgut cells of L. coleopterata have shallow infoldings of basal plasma membrane, and the cytoplasm is filled with mitochondria. Similar observations are found in the hindgut of
the G. maculata, indicating the hindgut is involved in ion reabsorption and the production of hypo-osmotic urine (Marshall and Cheung, 1973). However, no leaflets are observed in the ileum of L. coleopterata, although similar leaflets formed by invaginations of the apical plasma membrane are found in the ileum of the G. maculata. Concentric granules observed in the apical cytoplasm of the proximal Malpighian tubules of L. coleopterata (Fig. 4A, B) are very peculiar because they appear to contain a multilayer of laminae around the center, and each lamina has a different electron-density. They are morphologically similar to minerals-containing granules (especially calcium-containing granules) in alimentary canals of other arthropods previously investigated, which have been
H. Zhong et al. / Arthropod Structure & Development 42 (2013) 521e530
529
Fig. 7. (A) Detail of the cells of the posterior tubular midgut, showing elongated mitochondria (mi), secretory granules (sg), rough endoplasmic reticulum (rer), and nuclei (N) with evident nucleoli (n). (B) High magnification of the first cellular type of the posterior tubular midgut, showing aggregations of fine granules (asterisks) and mitochondria with cristae (arrows). (C) The second cellular type of the posterior tubular midgut features well-developed basal infoldings (if) and regularly-arranged microvilli (mv). Cytoplasm contains abundant mitochondria (mi), secretory vesicles (sv), nuclei (N) with small nucleoli (n), and scattered elements of rough endoplasmic reticulum (rer). (D) Detail of the second cellular type of posterior tubular midgut. Cells with well-developed basal infoldings (if) possess numerous mitochondria (mi), vesicles (v) filled with fine granular materials, and sparse rough endoplasmic reticulum (rer). (E) Transverse section of the distal tubular midgut. Cells possess well-developed basal infoldings (if) and dense microvilli (mv), and their cytoplasm is packed with mitochondria (mi). sd, septate desmosomes; bl, basal lamina; l, lumen; mu, muscles. Scale bars: 1.0 mm in (A, C, E); 0.5 mm in (B); 0.2 mm in (D).
reported to perform a skeletal function and a dynamic function, such as excretion, calcium mobilization, storage, and/or detoxication (Brown, 1982). But the composition and role of these granules need to be explored further. Microorganisms have been found in practically every arthropod alimentary canal examined, and some insects establish mutualistic symbiosis with microorganisms to meet their nutritional demands via synthesizing and supplying nutrients such as essential amino acids (Moran, 2007; Gündüz and Douglas, 2009). Insects of Cicadomorpha feed on plant phloem and/or xylem sap which provide an imbalanced diet containing rich sugars but poor nitrogen and essential amino acids (Terra, 1990). Therefore, they have adopted a
common solution to nutritional deficiencies, i.e., their nutritional source is related to widespread, mutualistic symbiotic bacteria in their alimentary canals and the associated Malpighian tubules. These organisms have the capacity to biosynthesize required amino acids (Douglas, 2006). In our investigation, microorganisms of different shapes are observed in the cytoplasm of the anterior extremity of the midgut, posterior extremity of the midgut, proximal Malpighian tubules and ileum of L. coleopterata (Figs. 3CeE, 8A); they are probably symbiotic to meet the demand for amino acids. Further work is needed to clarify their identity and functions, to provide more information for studies on the mutualism between L. coleopterata and related microorganisms.
530
H. Zhong et al. / Arthropod Structure & Development 42 (2013) 521e530
Fig. 8. Transverse section of the hindgut, showing a cuticle (c) lining the surface of the hindgut lumen. (A) The ileum cells contain numerous secretory vesicles (sv) and abundant mitochondria (mi). Double-membraned microorganisms (arrows) are visible among the secretory vesicles. (B) Cytoplasm of the rectum cell contains abundant mitochondria (mi). if, infoldings of basal plasma membrane; l, lumen; mu, muscles. Scale bars: 1.0 mm in (A, B).
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 study is supported by Program for New Century Excellent Talents in Universities (NCET10-0691) and the Key Project of Introducing Top Overseas Cultural & Educational Experts to the Northwest A&F University Founded by the Chinese Ministry of Education (Grant No. TS2011XBNL061). References Brown, B.E., 1982. The form and function of metal-containing ‘granules’ in invertebrate tissues. Biological Reviews 57, 621e667. Cecil, R., 1930. The alimentary canal of Philaenus leucophthalmus. Ohio Journal of Science 30, 120e130. Cheung, W.W.K., Marshall, A.T., 1973a. Studies on water and ion transport in homopteran insects: ultrastructure and cytochemistry of the cicadoid and cercopoid midgut. Tissue and Cell 5, 651e669. Cheung, W.W.K., Marshall, A.T., 1973b. Water and ion regulation in cicadas in relation to xylem feeding. Journal of Insect Physiology 19, 1801e1816. Cryan, J., Svenson, G., 2010. Family-level relationships of the spittlebugs and froghoppers (Hemiptera: Cicadomorpha: Cercopoidea). Systematic Entomology 35, 393e415. Dickson, R.E., 1979. Xylem translocation of amino acids from roots to shoots in cottonwood plants. Canadian Journal of Forest Research-revue Canadienne de Recherche Forestiere 9, 374e378. Douglas, A.E., 2006. Phloem-sap feeding by animals: problems and solutions. Journal of Experimental Botany 57, 747e754. Fonseca, F.V., Silva, J.R., Samuels, R.I., DaMatta, R.A., Terra, W.R., Silva, C.P., 2010. Purification and partial characterization of a midgut membrane-bound alphaglucosidase from Quesada gigas (Hemiptera: Cicadidae). Comparative Biochemistry and Physiology 155, 20e25. Gündüz, E.A., Douglas, A.E., 2009. Symbiotic bacteria enable insects to use a nutritionally inadequate diet. Proceedings of the Royal Society of London Series B 276, 987e991. Kershaw, J.C., 1914. The alimentary canal of a cercopid (Tomaspis saccarina Dist.). Psyche 21, 65e72.
Licent, P.E., 1912. Recherches d’anatomie et de physiologie comparees sur le tube digestif des Homopteres superieures. La Cellule 28, 6e161. Lindsay, K.L., Marshall, A.T., 1980. Ultrastructure of the filter chamber complex in the alimentary canal of Eurymela distincta Signoret (Homoptera, Eurymelidae). International Journal of Insect Morphology and Embryology 9, 179e198. Lindsay, K.L., Marshall, A.T., 1981. The osmoregulatory role of the filter chamber in relation to phloem-feeding in Eurymela distincta (Cicadelloidea, Homoptera). Physiological Entomology 6, 413e419. Marshall, A.T., Cheung, W.W.K., 1973. Studies on water and ion transport in homopteran insects: ultrastructure and cytochemistry of the cicadoid and cercopoid hindgut. Tissue and Cell 5, 671e678. Marshall, A.T., Cheung, W.W.K., 1974. Studies on water and ion transport in homopteran insects: ultrastructure and cytochemistry of the cicadoid and cercopoid Malpighian tubules and filter chamber. Tissue and Cell 6, 153e171. Mehrabadi, M., Bandani, A.R., 2011. Secretion and formation of perimicrovillar membrane in the digestive system of the sunn pest, Eurygaster integriceps (Hemiptera: Scutelleridae) in response to feeding. Archives of Insect Biochemistry and Physiology 78, 190e200. Moran, N.A., Tran, P., Gerardo, N.M., 2005. Symbiosis and insect diversification: an ancient symbiont of sap-feeding insects from the bacterial phylum Bacteroidetes. Applied and Environmental Microbiology 71, 8802e8810. Moran, N.A., 2007. Symbiosis as an adaptive process and source of phenotypic complexity. Proceedings of the National Academy of Sciences of the United States of America 104, 8627e8633. Silva, C.P., Ribeiro, A.F., Gulbenkian, S., Terra, W.R., 1995. Organization, origin and function of the outer microvillar (perimicrovillar) membranes of Dysdercus peruvianus (Hemiptera) midgut cells. Journal of Insect Physiology 41, 1093e 1103. Silva, C.P., Ribeiro, A.F., Terra, W.R., 1996. Enzyme markers and isolation of the microvillar and perimicrovillar membranes of Dysdercus peruvianus (Hemiptera: Pyrrhocoridae) midgut cells. Insect Biochemistry and Molecular Biology 26, 1011e1018. Silva, C.P., Silva, J.R., Vasconcelos, F.F., Petretski, M.D.A., DaMatta, R.A., 2004. Occurrence of midgut perimicrovillar membranes in paraneopteran insect orders with comments on their function and evolutionary significance. Arthropod Structure and Development 33, 139e148. Terra, W.R., 1988. Physiology and biochemistry of insect digestion: an evolutionary perspective. Brazilian Journal of Medical and Biological Research 21, 675e734. Terra, W.R., 1990. Evolution of digestive systems of insects. Annual Review of Entomology 35, 181e200. Zhong, H.Y., Wei, C., Wang, Y., Li, Q.L., Zhao, Y., Wei, L.Y., 2011. The morphological and functional differentiation of the legs, alimentary canals and Malpighian tubules of Cicadomorpha, and its implications to the phylogeny of Cicadomorpha. Acta Zootaxonomica Sinica 36, 670e680.
Contents lists available at SciVerse ScienceDirect
Arthropod Structure & Development journal homepage: www.elsevier.com/locate/asd
Anatomy and fine structure of the alimentary canal of the spittlebug Lepyronia coleopterata (L.) (Hemiptera: Cercopoidea) Haiying Zhong, Yalin Zhang*, Cong Wei* Key Laboratory of Plant Protection Resources and Pest Management, Ministry of Education, Entomological Museum, Northwest A&F University, Yangling, Shaanxi 712100, China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 15 March 2013 Accepted 30 April 2013
The alimentary canal of the spittlebug Lepyronia coleopterata (L.) differentiates into esophagus, filter chamber, midgut (conical segment, tubular midgut), and hindgut (ileum, rectum). The filter chamber is composed of the anterior extremity of the midgut, posterior extremity of the midgut, proximal Malpighian tubules, and proximal ileum; it is externally enveloped by a thin cellular sheath and thick muscle layers. The sac-like anterior extremity of the midgut is coiled around by the posterior extremity of the midgut and proximal Malpighian tubules. The tubular midgut is subdivided into an anterior tubular midgut, midmidgut, posterior tubular midgut, and distal tubular midgut. Four Malpighian tubules run alongside the ileum, and each terminates in a rod closely attached to the rectum. Ultrastructurally, the esophagus is lined with a cuticle and enveloped by circular muscles; its cytoplasm contains virus-like fine granules of high electron-density. The anterior extremity of the midgut consists of two cellular types: (1) thin epithelia with well-developed and regularly arranged microvilli, and (2) large cuboidal cells with short and sparse microvilli. Cells of the posterior extremity of the midgut have regularly arranged microvilli and shallow basal infoldings devoid of mitochondria. Cells of the proximal Malpighian tubule possess concentric granules of different electron-density. The internal proximal ileum lined with a cuticle facing the lumen and contains secretory vesicles in its cytoplasm. Dense and long microvilli at the apical border of the conical segment cells are coated with abundant electron-dense fine granules. Cells of the anterior tubular midgut contain spherical secretory granules, oval secretory vesicles of different size, and autophagic vacuoles. Ferritin-like granules exist in the mid-midgut cells. The posterior tubular midgut consists of two cellular types: 1) cells with shallow and bulb-shaped basal infoldings containing numerous mitochondria, homocentric secretory granules, and fine electron-dense granules, and 2) cells with well-developed basal infoldings and regularly-arranged apical microvilli containing vesicles filled with fine granular materials. Cells of the distal tubular midgut are similar to those of the conical segment, but lack electron-dense fine granules coating the microvilli apex. Filamentous materials coat the microvilli of the conical segment, anterior and posterior extremities of the midgut, which are possibly the perimicrovillar membrane closely related to the nutrient absorption. The lumen of the hindgut is lined with a cuticle, beneath which are cells with poorly-developed infoldings possessing numerous mitochondria. Single-membraned or doublemembraned microorganisms exist in the anterior and posterior extremities of the midgut, proximal Malpighian tubule and ileum; these are probably symbiotic. Ó 2013 Elsevier Ltd. All rights reserved.
Keywords: Alimentary tract Morphology Ultrastructure Auchenorrhyncha Aphrophoridae
1. Introduction As the second most species-rich superfamily in the Cicadomorpha, Cercopoidea includes approximately 3000 described species classified into five families: Cercopidae, Aphrophoridae,
* Corresponding authors. Tel./fax: þ86 29 87092509. E-mail addresses: [email protected] (H. Zhong), [email protected] (Y. Zhang), [email protected] (C. Wei). 1467-8039/$ e see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.asd.2013.04.005
Clastopteridae, Machaerotidae and Epipygidae (Cryan and Svenson, 2010). They are commonly called spittlebugs or froghoppers, and the former name is attributed to the nymphs immersing their soft bodies in a frothy saliva-like mass that protects them from desiccation and predators. Spittlebugs feed exclusively on xylem sap, which is an imbalanced diet and has a low concentration of nutrients (Cheung and Marshall, 1973b; Dickson, 1979). In order to eliminate excessive water from the ingested juice and concentrate the nutrients effectively, part of their alimentary canal modifies into a junction
522
H. Zhong et al. / Arthropod Structure & Development 42 (2013) 521e530
known as “filter chamber”, which consists of the anterior and posterior extremities of the midgut as well as the proximal Malpighian tubules (Marshall and Cheung, 1974). The filter chamber is a shunting complex that can concentrate ingested xylem fluid approximately 10-fold (Cheung and Marshall, 1973b) and phloem sap about 2.5-fold (Lindsay and Marshall, 1981); excessive water can be directly transferred from the anterior extremity of the midgut to the hindgut (Marshall and Cheung, 1974). In addition, the alimentary canal may contain endosymbionts which make a significant contribution to their hosts’ nutrient absorption (Moran et al., 2005). The alimentary canal of spittlebugs has been morphologically and/or histologically investigated for only a limited number of species (Kershaw, 1914; Cecil, 1930; Cheung and Marshall, 1973a; Marshall and Cheung, 1973, 1974). Although these studies have provided important data about the fine structure and cytochemistry of the alimentary canal in those species, more detailed information on the alimentary canal of other species is still needed, and the location of microorganisms that reside either in the lumen or in the cytoplasm of the alimentary canal also merits further investigation. Herein, we investigate the gross morphology and fine structure of the alimentary canal of Lepyronia coleopterata (Linnaeus), a widely distributed species in China, using both light microscopy and transmission electron microscopy. We are aiming to elucidate the alimentary canal at the morphological and ultrastructural levels, and to provide more information for future investigation about the microorganisms that reside in the cytoplasm.
a slender and elongate esophagus (Oe) that runs posteriorly and connects with the filter chamber (FC). The filter chamber is a complex junction with four, clearly defined regions: anterior extremity of the midgut, posterior extremity of the midgut, proximal Malpighian tubules, and proximal ileum. The greatly expanded anterior extremity of the midgut is coiled around by the slender posterior extremity of the midgut and proximal Malpighian tubules. A very thin cellular sheath and thick muscle layers externally enclose the filter chamber. The midgut outside the filter chamber is differentiated into two distinct regions: a considerably inflated saclike conical segment (CS), and a very slender tubular midgut. The tubular midgut has been divided into four regions based on their morphological and ultrastructural characters: a thin anterior tubular midgut (AMg), a very thick mid-midgut (MMg), a thick posterior tubular midgut (PMg), and a thin distal tubular midgut (DMg). It exhibits a “U-shaped” path as follows: the anterior tubular midgut connecting with the conical segment descends backwards and connects with the mid-midgut, which runs back to the posterior tubular midgut; the posterior tubular midgut ascends and connects to the distal tubular midgut entering into the filter chamber. The hindgut emerges halfway along the filter chamber and consists of a long and uniform ileum (Il) and a slightly inflated rectum (Rc). Four Malpighian tubules (MT) run alongside the ileum, and each of them terminates in a rod closely attached to the rectum (Fig. 1).
2. Materials and methods
3.2.1. Esophagus The esophagus is externally enveloped by well-developed circular muscles and internally is lined with a layer of convoluted cuticle (about 0.1 mm) (Fig. 2A). Beneath the cuticle are epithelia resting on a basal lamina (about 2.0 mm thick) (Fig. 2A). The basal plasma membrane of the epithelium invaginates into poorly developed infoldings. In the cytoplasm, mitochondria, elongated nuclei and fine virus-like granules (about 0.07 mm in diameter) of high electron-density are observed (Fig. 2B).
Specimens of male L. coleopterata were collected in Yangling, Shaanxi Province, China, in June 2010 and 2012. 2.1. Light microscopy The live L. coleopterata were anesthetized by chilling and their alimentary canals were dissected out in phosphate buffer (PBS, 0.2 M, pH 7.2) under a Motic SMZ168 Stereoscopic Zoom Microscope. Photographs were taken with a scientific digital micrography system equipped with an Auto-montage imaging system and a Qimaging Retiga 2000R digital camera (CCD).
3.2. Ultrastructure of the alimentary canal
3.2.2. Filter chamber Ultrastructurally, different regions of the filter chamber exhibit significant differences, and their typical characteristics are described in detail below.
2.2. Transmission electron microscopy The dissected alimentary canals were pre-fixed in 2.5% glutaraldehyde (phosphate buffer, 0.2 M, pH 7.2) at 4 C for 12 h, washed five times with 0.1 M phosphate buffer (pH 7.2) and post-fixed in 1% osmium tetroxide for 2 h at 4 C. Then the materials were rinsed five times in the same phosphate buffer (0.1 M, pH 7.2). After dehydration in an ethanol series and 100% acetone and infiltration with a mixture of Epon 812 in acetone (1: 3 (v/v) for 2 h, 1: 1 (v/v) for 5 h, 3: 1 (v/v) for 12 h, separately), the samples were embedded in pure Epon 812 and incubated at 30 C for 24 h, at 60 C for 48 h, respectively. Ultra-thin sections were stained with uranyl acetate and lead citrate, and examined under a JEOL JEM-1230 TEM at 80 kV and photographed. The terminology of the alimentary canal structures follows that of Cheung and Marshall (1973a) and Marshall and Cheung (1974). 3. Results 3.1. Gross morphology of the alimentary canal The alimentary canal of L. coleopterata consists of esophagus, filter chamber, midgut, and hindgut. The alimentary tract begins as
3.2.3. Cellular sheath The thin cellular sheath comprising numerous long and thin cells forms a recticulum-shaped structure. The sheath cells are externally surrounded by well-developed circular muscles with large nuclei and rod nucleoli (Fig. 2C). 3.2.4. Anterior extremity of midgut (the filter chamber proper) The anterior extremity of the midgut consists of two types of cells: thin epithelium, and large cuboidal cells. Both types of cells lie on a thin basal membrane and display great differences at the ultrastructural level. The thin epithelium has the following characters: 1) welldeveloped and regularly-arranged apical microvilli are coated by filamentous materials; 2) basal plasma membrane invaginates into regularly spaced and tubular infoldings; and 3) the cytoplasm is packed with abundant mitochondria (Fig. 2D). The large cuboidal cells feature very short and sparse microvilli at the apical border, and secretory vesicles, secretory granules, mitochondria and rough endoplasmic reticulum exist in the cytoplasm (Fig. 3A). These secretory granules differ in size, shape and electron-density, with some electron-lucent granules containing radial inclusions of high electron-density (Fig. 3A); some secretory
H. Zhong et al. / Arthropod Structure & Development 42 (2013) 521e530
523
Fig. 1. General structure of alimentary canal of Lepyronia coleopterata (L.), with one Malpighian tubule left and the other three tubules removed. AMg, anterior tubular midgut; CS, conical segment; DMg, distal tubular midgut; FC, filter chamber; Il, ileum; MMg, mid-midgut; MT, Malpighian tubule; Oe, esophagus; PMg, posterior tubular midgut; Rc, rectum.
granules are electron-dense in the center and electron-lucent at the periphery (Fig. 3B). Septate desmosomes joining the large cuboidal cells are also visible in the cytoplasm (Figs. 3A, B). A cluster of intracellular single-membraned microorganisms (about 0.25e 0.40 mm in diameter) is observed adjacent to the rough endoplasmic reticulum (Fig. 3C). 3.2.5. Posterior extremity of the midgut Regularly-arranged apical microvilli coated by filamentous materials and wide basal infoldings devoid of mitochondria are characteristics of cells in this gut region. Scattered elements of rough endoplasmic reticulum are observed in the cytoplasm, where lysosome-like structures and double-membraned microorganisms are also contained (Fig. 3D). 3.2.6. Proximal Malpighian tubule (internal Malpighian tubule) The proximal Malpighian tubule consists of a single layer of cells with well-developed and regularly-arranged basal infoldings and apical microvilli (Figs. 2C, 3E and 4A). Double-membraned microorganisms and scattered rough endoplasmic reticulum are observed in the cytoplasm (Fig. 3E). At the apical region of the cell, concentric granules (about 2.0 mm in diameter) are prominent with electron-dense gradations from the center to periphery (Fig. 4A). Some concentric granules display filamentous-like areas in their peripheries (Fig. 4B). Large nuclei with obvious nucleoli exist in the cytoplasm (Fig. 4C). 3.2.7. Internal proximal ileum The internal proximal ileum is lined with a layer of cuticle facing the lumen. Beneath the cuticle are cells containing secretory
vesicles and large nuclei. Tracheoles penetrate the cells and even reach to the cytoplasm (Fig. 4D). 3.2.8. Conical segment Cells of the conical segment rest on a basal lamina, outside of which are well-developed circular muscles (Fig. 5A). Their basal plasma membrane invaginates into well-developed infoldings extending deeply into rough endoplasmic reticulum (Fig. 5B). Mitochondria of different shape are visible around the scattered elements of rough endoplasmic reticulum (Fig. 5B). The apical border of the cell bears long and dense microvilli coated by filamentous materials and aggregations of electron-dense fine granules (Fig. 5C). 3.2.9. Anterior tubular midgut (AMg) The anterior tubular midgut is externally surrounded by conspicuous longitudinal muscles (Fig. 5D). Its cells lie on a thin basal lamina, and are characterized by well-developed basal infoldings associated with mitochondria and dense, regularly-arranged apical microvilli (Fig. 5D). Spherical secretory granules and oval secretory vesicles of different sizes are observed at the basal area of the cells. Mitochondria, sparse rough endoplasmic reticulum, large autophagic vacuoles and small vacuoles exist in the cytoplasm (Figs. 5D and 6A). 3.2.10. Mid-midgut (MMg) Cells of the mid-midgut also lie on a thin basal lamina. At the basal region, their basal plasma membrane invaginates into welldeveloped infoldings associated with mitochondria (Fig. 6B). Rough endoplasmic reticulum is scattered through the cytoplasm,
524
H. Zhong et al. / Arthropod Structure & Development 42 (2013) 521e530
Fig. 2. (A) Transverse section of esophagus lined with a convoluted cuticle (c) facing the lumen (l), thick basal lamina (bl) and basal infoldings (if) are also visible. (B) Detail of the cytoplasm of the esophagus, with aggregations of virus-like fine granules (white asterisks), a thick cuticle (c) and elongate nuclei (N). Secretions (black asterisks) exist in the lumen. (C) Cells of cellular sheath (cs), which are externally surrounded by well-developed circular muscles (mu) containing nuclei (N) with evident nucleoli (n). Basal infoldings (if) and apical microvilli (mv) of the cells of the proximal Malpighian tubule (imt) (arrow) are also obvious. (D) Transverse section of the anterior extremity of the midgut. Filamentous materials (fm) coat the apical microvilli (mv) facing the lumen (l). Basal infoldings (if) are tubular-shaped and regularly-spaced. Abundant mitochondria (mi) exist throughout the cytoplasm. bl, basal lamina. Scale bars: 5.0 mm in (A); 0.5 mm in (B); 2.0 mm in (C, D).
among which are numerous ferritin-like granules, mitochondria, and nuclei (Fig. 6C). The apical microvilli are less dense than those of the anterior tubular midgut (Figs. 5D and 6A, C). 3.2.11. Posterior tubular midgut (PMg) Cells of the posterior tubular midgut are significantly different from cells of other gut regions, and are categorized into two distinct types. The first type of cells is characterized by very shallow and bulb-shaped basal infoldings, and their cytoplasm is packed with secretory granules and mitochondria (Fig. 6D). These secretory granules vary in size and shape, and smaller granules appear to fuse to become larger ones (Figs. 6D and 7A). Scattered elements of rough endoplasmic reticulum and polyploid nuclei with evident
nucleoli are also visible among the secretory granules (Fig. 7A). In the cytoplasm, aggregations of fine electron-dense granules and mitochondria with cristae are observed (Fig. 7B). Compared with the first type of cells, the second type of cells has the following features: their basal infoldings are well-developed and associated with mitochondria; apical microvilli are dense and regularly arranged; large nuclei with evident nucleoli are surrounded by numerous mitochondria, sparse rough endoplasmic reticulum, and secretory vesicles filled with fine granular materials (Fig. 7C, D). 3.2.12. Distal tubular midgut (DMg) Cells of the distal tubular midgut are similar to those of the conical segment, except they lack filamentous materials and
H. Zhong et al. / Arthropod Structure & Development 42 (2013) 521e530
525
Fig. 3. (A) The apical region of the large cuboidal cells of the anterior extremity of the midgut (filter chamber proper). Rough endoplasmic reticulum (rer), septate desmosomes (sd), mitochondria (mi), sparse microvilli (mv), secretory vesicles (sv), and electron-dense secretory granules (sg) are visible. (B) Cytoplasm of the large cuboidal cells of the anterior extremity of midgut (filter chamber proper) contains extensive rough endoplasmic reticulum (rer) and secretory granules (sg) of electron-dense center and electron-lucent periphery. (C) Large cuboidal cells of the anterior extremity midgut (filter chamber proper), showing extensive rough endoplasmic reticulum (rer), electron-dense secretory granules (sg) and microorganisms (arrows). (D) Transverse section of the posterior extremity of the midgut. Regularly-arranged microvilli (mv) are evident. Filamentous materials (fm) cover the apex of apical microvilli. Basal plasma membrane invaginates into wide infoldings (if). The cytoplasm contains microorganisms (arrow), lysosome-like structures (ly) and rough endoplasmic reticulum (rer). (E) Transverse section of the proximal Malpighian tubule, showing well-developed and regularly-arranged basal infoldings (if) and apical microvilli (mv). Scattered rough endoplasmic reticulum (rer) and microorganisms (arrows) are also seen in the cytoplasm. bl, basal lamina; l, lumen; Scale bars: 0.5 mm in (A, B, C); 0.2 mm in (D, E).
electron-dense fine granules covering the apex of microvilli (Fig. 7E). 3.2.13. Hindgut The hindgut contains two parts (ileum and rectum) and is lined with a cuticle facing the lumen. Cells underlying the cuticle possess very poorly developed basal infoldings, while abundant mitochondria exist within the cytoplasm. However, some ultrastructural differences also exist between the ileum and rectum. At the basal area of the ileum cells, double-membraned microorganisms and electron-lucent secretory vesicles of different sizes are observed, and the smaller secretory vesicles seem to fuse to become larger
ones (Fig. 8A). The microorganisms are morphologically similar to those found in the cytoplasm of the posterior extremity of the midgut and the proximal Malpighian tubules, but differ greatly from those detected in the cells of the anterior extremity of the midgut where single-membraned microorganisms reside (Fig. 3C). In the cells of the rectum, however, no elements such as secretory vesicles and microorganisms are observed (Fig. 8B). 4. Discussion The general structure of the alimentary canal of L. coleopterata (L.) observed in this investigation closely resembles that depicted
526
H. Zhong et al. / Arthropod Structure & Development 42 (2013) 521e530
Fig. 4. (A) Transverse section of the proximal Malpighian tubule, showing concentric granules (cg) with different electron-density from the center to the periphery. (B). Apical area of the cells of the proximal Malpighian tubule. Concentric granules (cg) with filamentous periphery are shown. (C) Cells of the proximal Malpighian tubule contain large nuclei (N) with evident nucleoli (n). The apical microvilli (mv) are dense and long. (D) The internal proximal ileum is lined with a layer of cuticle (c) facing the lumen (l), and its cytoplasm contains large nuclei (N) and secretory vesicles (sv). Tracheoles (tr) are well-developed and penetrate into the plasma membrane. bl, basal lamina; if, infoldings of the basal plasma membrane; mi, mitochondria. Scale bars: 1.0 mm in (A, C); 0.5 mm in (B); 2.0 mm in (D).
in previous studies of other spittlebug, leafhopper and cicada species (Licent, 1912; Kershaw, 1914; Cecil, 1930; Cheung and Marshall, 1973a; Marshall and Cheung, 1973, 1974; Zhong et al., 2011). The canal is differentiated into at least four clearly defined regions: an elongate esophagus, a modified filter chamber, a midgut (including a sac-like conical segment and a tubular midgut), and a hindgut. Recently, the perimicrovillar membrane, a lipoprotein membrane, was detected in the midgut cells of some hemipterans, e.g., the spittlebug Mahanarva posticata, the cicada Quesada gigas and the bug Eurygaster integriceps, which coats the microvilli from their base to apex and extends into the gut lumen (Silva et al., 1995, 1996, 2004; Terra, 1988; Fonseca et al., 2010; Mehrabadi and Bandani, 2011). The perimicrovillar membrane exhibits a
function similar to the peritrophic matrix of the midgut as in some other insects with chewing mouthparts (Terra, 1988). Our fine structure investigation found that filamentous materials coat the apical microvilli of cells of the anterior extremity of the midgut, the posterior extremity of the midgut, as well as the conical segment of L. coleopterata (Figs. 2D, 3D and 5C). Similar materials, previously termed “filamentous coats”, have been described as covering the surface or apex of the apical microvilli of the filter chamber proper and midgut of some other species in Cicadomorpha (Cheung and Marshall, 1973a; Marshall and Cheung, 1974; Lindsay and Marshall, 1980). Marshall and Cheung (1974) deduced that such coatings probably function to bind cations (especially potassium) contained in the xylem sap. Filamentous materials observed in our study are possibly the
H. Zhong et al. / Arthropod Structure & Development 42 (2013) 521e530
527
Fig. 5. (A) Transverse section of the conical segment, showing well-developed circular muscles (mu) that envelope the cells, which have well-developed basal infoldings (if) and dense microvilli (mv). Nuclei (N) and abundant mitochondria (mi) are observed in the cytoplasm. (B) High magnification of the basal area of the conical segment cells. Basal plasma membrane invaginates into wide infoldings (if) extending to scattered rough endoplasmic reticulum (rer). Mitochondria (mi) of different shapes exist among the rough endoplasmic reticulum. (C) High magnification of the apical region of the conical segment cells. Filamentous materials (fm) and abundant electron-dense fine granules (asterisks) cover the apex of apical microvilli (mv). (D) Transverse section of the anterior tubular midgut. Cells with well-developed basal infoldings (if) contain abundant mitochondria (mi). Secretory vesicles (sv) and secretory granules (sg) are also evident in the cytoplasm. Microvilli (mv) are dense and regularly-arranged. Nuclei (N) and rough endoplasmic reticulum (rer) scatter in the cytoplasm. l, lumen; mu, muscles; bl, basal lamina; sd, septate desmosomes. Scale bars: 2.0 mm in (A, D); 0.5 mm in (B); 1.0 mm in (C).
perimicrovillar membrane, similar to their morphology; such a membrane should be closely related to nutrient absorption, as in other fluid-feeding hemipterans. Special fine granules of high electron-density are observed in the conical segment cells of the alimentary canal of L. coleopterata (Fig. 5C), which have never been reported for hemipterans, to date. The existence of these granules at the apex of the microvilli of the conical segment suggests that those granules are associated with food digestion and nutrient absorption, since the conical segment has been reported to be one of the regions for nutrient absorption (Cheung and Marshall, 1973a). In addition, secretory granules and/or vesicles are also found in cells of different gut
regions of L. coleopterata, such as the anterior extremity of the midgut, internal proximal ileum, anterior tubular midgut, posterior tubular midgut and ileum (Figs. 3AeC, 4D, 5D, 6D, 7A, C, 8A), indicating that these regions play a vital role in secretion. However, the nature and function of these fine granules needs to be investigated further. Basal infoldings of cells in the anterior extremity of the midgut (filter chamber proper) of L. coleopterata are tubular-shaped and regularly-spaced (Fig. 2D). They are structurally similar to depictions for the cicada Gaeana maculata by Marshall and Cheung (1974), and were speculated to possess an intrinsic rigidity to prevent them from collapsing during contraction of the filter
528
H. Zhong et al. / Arthropod Structure & Development 42 (2013) 521e530
Fig. 6. Electron micrographs of the tubular midgut. (A) Detail of the apical region of the cells of anterior tubular midgut, showing autophagic vacuoles (av) and small vacuoles (v). The cytoplasm contains abundant mitochondria (mi) and scattered elements of rough endoplasmic reticulum (rer). Long apical microvilli (mv) are regularlyarranged and dense. (B) Basal region of mid-midgut cells, showing well-developed basal infoldings (if) associated with mitochondria (mi). (C) Apical parts of the cells of the mid-midgut. The cytoplasm contains ferritin-like granules (f), mitochondria (mi) and sparse rough endoplasmic reticula (rer). The apical microvilli are long but sparse, and some of them are ruptured. (D) The first cellular type of the posterior tubular midgut. Shallow and bulb-shaped basal infoldings (if) are visible. Cytoplasm filled with secretory granules (sg) and mitochondria (mi). The secretory granules are electron-lucent in center and electron-dense at periphery. bl, basal lamina; N, nuclei. Scale bars: 1.0 mm in (A, B, C); 2.0 mm in (D).
chamber (Marshall and Cheung, 1974). However, basal infoldings found in the cells of other gut regions of L. coleopterata are morphologically different from the above-mentioned infoldings, viz., they are not tubular shaped and irregularly-spaced. In addition, basal infoldings found in the gut cells other than in the anterior extremity of midgut (filter chamber proper) are associated with mitochondria. This probably indicates that these cells perform a strong secretory function, as has been suggested for pylorus cells in the leafhopper Eurymela distincta (Lindsay and Marshall, 1980). The hindgut cells of L. coleopterata have shallow infoldings of basal plasma membrane, and the cytoplasm is filled with mitochondria. Similar observations are found in the hindgut of
the G. maculata, indicating the hindgut is involved in ion reabsorption and the production of hypo-osmotic urine (Marshall and Cheung, 1973). However, no leaflets are observed in the ileum of L. coleopterata, although similar leaflets formed by invaginations of the apical plasma membrane are found in the ileum of the G. maculata. Concentric granules observed in the apical cytoplasm of the proximal Malpighian tubules of L. coleopterata (Fig. 4A, B) are very peculiar because they appear to contain a multilayer of laminae around the center, and each lamina has a different electron-density. They are morphologically similar to minerals-containing granules (especially calcium-containing granules) in alimentary canals of other arthropods previously investigated, which have been
H. Zhong et al. / Arthropod Structure & Development 42 (2013) 521e530
529
Fig. 7. (A) Detail of the cells of the posterior tubular midgut, showing elongated mitochondria (mi), secretory granules (sg), rough endoplasmic reticulum (rer), and nuclei (N) with evident nucleoli (n). (B) High magnification of the first cellular type of the posterior tubular midgut, showing aggregations of fine granules (asterisks) and mitochondria with cristae (arrows). (C) The second cellular type of the posterior tubular midgut features well-developed basal infoldings (if) and regularly-arranged microvilli (mv). Cytoplasm contains abundant mitochondria (mi), secretory vesicles (sv), nuclei (N) with small nucleoli (n), and scattered elements of rough endoplasmic reticulum (rer). (D) Detail of the second cellular type of posterior tubular midgut. Cells with well-developed basal infoldings (if) possess numerous mitochondria (mi), vesicles (v) filled with fine granular materials, and sparse rough endoplasmic reticulum (rer). (E) Transverse section of the distal tubular midgut. Cells possess well-developed basal infoldings (if) and dense microvilli (mv), and their cytoplasm is packed with mitochondria (mi). sd, septate desmosomes; bl, basal lamina; l, lumen; mu, muscles. Scale bars: 1.0 mm in (A, C, E); 0.5 mm in (B); 0.2 mm in (D).
reported to perform a skeletal function and a dynamic function, such as excretion, calcium mobilization, storage, and/or detoxication (Brown, 1982). But the composition and role of these granules need to be explored further. Microorganisms have been found in practically every arthropod alimentary canal examined, and some insects establish mutualistic symbiosis with microorganisms to meet their nutritional demands via synthesizing and supplying nutrients such as essential amino acids (Moran, 2007; Gündüz and Douglas, 2009). Insects of Cicadomorpha feed on plant phloem and/or xylem sap which provide an imbalanced diet containing rich sugars but poor nitrogen and essential amino acids (Terra, 1990). Therefore, they have adopted a
common solution to nutritional deficiencies, i.e., their nutritional source is related to widespread, mutualistic symbiotic bacteria in their alimentary canals and the associated Malpighian tubules. These organisms have the capacity to biosynthesize required amino acids (Douglas, 2006). In our investigation, microorganisms of different shapes are observed in the cytoplasm of the anterior extremity of the midgut, posterior extremity of the midgut, proximal Malpighian tubules and ileum of L. coleopterata (Figs. 3CeE, 8A); they are probably symbiotic to meet the demand for amino acids. Further work is needed to clarify their identity and functions, to provide more information for studies on the mutualism between L. coleopterata and related microorganisms.
530
H. Zhong et al. / Arthropod Structure & Development 42 (2013) 521e530
Fig. 8. Transverse section of the hindgut, showing a cuticle (c) lining the surface of the hindgut lumen. (A) The ileum cells contain numerous secretory vesicles (sv) and abundant mitochondria (mi). Double-membraned microorganisms (arrows) are visible among the secretory vesicles. (B) Cytoplasm of the rectum cell contains abundant mitochondria (mi). if, infoldings of basal plasma membrane; l, lumen; mu, muscles. Scale bars: 1.0 mm in (A, B).
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 study is supported by Program for New Century Excellent Talents in Universities (NCET10-0691) and the Key Project of Introducing Top Overseas Cultural & Educational Experts to the Northwest A&F University Founded by the Chinese Ministry of Education (Grant No. TS2011XBNL061). References Brown, B.E., 1982. The form and function of metal-containing ‘granules’ in invertebrate tissues. Biological Reviews 57, 621e667. Cecil, R., 1930. The alimentary canal of Philaenus leucophthalmus. Ohio Journal of Science 30, 120e130. Cheung, W.W.K., Marshall, A.T., 1973a. Studies on water and ion transport in homopteran insects: ultrastructure and cytochemistry of the cicadoid and cercopoid midgut. Tissue and Cell 5, 651e669. Cheung, W.W.K., Marshall, A.T., 1973b. Water and ion regulation in cicadas in relation to xylem feeding. Journal of Insect Physiology 19, 1801e1816. Cryan, J., Svenson, G., 2010. Family-level relationships of the spittlebugs and froghoppers (Hemiptera: Cicadomorpha: Cercopoidea). Systematic Entomology 35, 393e415. Dickson, R.E., 1979. Xylem translocation of amino acids from roots to shoots in cottonwood plants. Canadian Journal of Forest Research-revue Canadienne de Recherche Forestiere 9, 374e378. Douglas, A.E., 2006. Phloem-sap feeding by animals: problems and solutions. Journal of Experimental Botany 57, 747e754. Fonseca, F.V., Silva, J.R., Samuels, R.I., DaMatta, R.A., Terra, W.R., Silva, C.P., 2010. Purification and partial characterization of a midgut membrane-bound alphaglucosidase from Quesada gigas (Hemiptera: Cicadidae). Comparative Biochemistry and Physiology 155, 20e25. Gündüz, E.A., Douglas, A.E., 2009. Symbiotic bacteria enable insects to use a nutritionally inadequate diet. Proceedings of the Royal Society of London Series B 276, 987e991. Kershaw, J.C., 1914. The alimentary canal of a cercopid (Tomaspis saccarina Dist.). Psyche 21, 65e72.
Licent, P.E., 1912. Recherches d’anatomie et de physiologie comparees sur le tube digestif des Homopteres superieures. La Cellule 28, 6e161. Lindsay, K.L., Marshall, A.T., 1980. Ultrastructure of the filter chamber complex in the alimentary canal of Eurymela distincta Signoret (Homoptera, Eurymelidae). International Journal of Insect Morphology and Embryology 9, 179e198. Lindsay, K.L., Marshall, A.T., 1981. The osmoregulatory role of the filter chamber in relation to phloem-feeding in Eurymela distincta (Cicadelloidea, Homoptera). Physiological Entomology 6, 413e419. Marshall, A.T., Cheung, W.W.K., 1973. Studies on water and ion transport in homopteran insects: ultrastructure and cytochemistry of the cicadoid and cercopoid hindgut. Tissue and Cell 5, 671e678. Marshall, A.T., Cheung, W.W.K., 1974. Studies on water and ion transport in homopteran insects: ultrastructure and cytochemistry of the cicadoid and cercopoid Malpighian tubules and filter chamber. Tissue and Cell 6, 153e171. Mehrabadi, M., Bandani, A.R., 2011. Secretion and formation of perimicrovillar membrane in the digestive system of the sunn pest, Eurygaster integriceps (Hemiptera: Scutelleridae) in response to feeding. Archives of Insect Biochemistry and Physiology 78, 190e200. Moran, N.A., Tran, P., Gerardo, N.M., 2005. Symbiosis and insect diversification: an ancient symbiont of sap-feeding insects from the bacterial phylum Bacteroidetes. Applied and Environmental Microbiology 71, 8802e8810. Moran, N.A., 2007. Symbiosis as an adaptive process and source of phenotypic complexity. Proceedings of the National Academy of Sciences of the United States of America 104, 8627e8633. Silva, C.P., Ribeiro, A.F., Gulbenkian, S., Terra, W.R., 1995. Organization, origin and function of the outer microvillar (perimicrovillar) membranes of Dysdercus peruvianus (Hemiptera) midgut cells. Journal of Insect Physiology 41, 1093e 1103. Silva, C.P., Ribeiro, A.F., Terra, W.R., 1996. Enzyme markers and isolation of the microvillar and perimicrovillar membranes of Dysdercus peruvianus (Hemiptera: Pyrrhocoridae) midgut cells. Insect Biochemistry and Molecular Biology 26, 1011e1018. Silva, C.P., Silva, J.R., Vasconcelos, F.F., Petretski, M.D.A., DaMatta, R.A., 2004. Occurrence of midgut perimicrovillar membranes in paraneopteran insect orders with comments on their function and evolutionary significance. Arthropod Structure and Development 33, 139e148. Terra, W.R., 1988. Physiology and biochemistry of insect digestion: an evolutionary perspective. Brazilian Journal of Medical and Biological Research 21, 675e734. Terra, W.R., 1990. Evolution of digestive systems of insects. Annual Review of Entomology 35, 181e200. Zhong, H.Y., Wei, C., Wang, Y., Li, Q.L., Zhao, Y., Wei, L.Y., 2011. The morphological and functional differentiation of the legs, alimentary canals and Malpighian tubules of Cicadomorpha, and its implications to the phylogeny of Cicadomorpha. Acta Zootaxonomica Sinica 36, 670e680.