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Arthropod Structure & Development 38 (2009) 377–389

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Arthropod Structure & Development

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The larval alimentary canal of the Antarctic insect, Belgica antarctica

James B. Nardi a,*, Lou Ann Miller b, Charles Mark Bee c, Richard E. Lee, Jr. d, David L. Denlinger e a Department of Entomology, University of Illinois, 320 Morrill Hall, 505 S. Goodwin Avenue, Urbana, IL 61801, USA b Center for Microscopy and Imaging, College of Veterinary Medicine, University of Illinois, 2001 S. Lincoln Avenue, Urbana, IL 61801, USA c Imaging Technology Group, Beckman Institute for Advanced Science and Technology, University of Illinois, 405N Mathews Avenue, Urbana, IL 61801, USA d Department of Zoology, Miami University, Oxford, OH 45056, USA e Department of Entomology, Ohio State University, 400 Aronoff Laboratory, 318 West 12th Avenue, Columbus, OH 43210, USA article info abstract

Article history: On the Antarctica continent the wingless midge, Belgica antarctica (Diptera, Chironomidae) occurs Received 26 October 2008 further south than any other insect. The digestive tract of the larval stage of Belgica that inhabits this Accepted 17 April 2009 extreme environment and feeds in detritus of penguin rookeries has been described for the ﬁrst time. Ingested food passes through a foregut lumen and into a stomodeal valve representing an intussus- Keywords: ception of the foregut into the midgut. A sharp discontinuity in microvillar length occurs at an interface Alimentary canal separating relatively long microvilli of the stomodeal midgut region, the site where peritrophic Midge membrane originates, from the midgut epithelium lying posterior to this stomodeal region. Although Antarctica Stomodeal valve shapes of cells along the length of this non-stomodeal midgut epithelium are similar, the lengths of their Microvilli microvilli increase over two orders of magnitude from anterior midgut to posterior midgut. Infoldings of Regenerative cells the basal membranes also account for a greatly expanded interface between midgut cells and the hemocoel. The epithelial cells of the hindgut seem to be specialized for exchange of water with their environment, with the anterior two-thirds of the hindgut showing highly convoluted luminal membranes and the posterior third having a highly convoluted basal surface. The lumen of the middle third of the hindgut has a dense population of resident bacteria. Regenerative cells are scattered throughout the larval midgut epithelium. These presumably represent stem cells for the adult midgut, while a ring of cells, marked by a discontinuity in nuclear size at the midgut-hindgut interface, presumably represents stem cells for the adult hindgut. Ó 2009 Elsevier Ltd. All rights reserved.

1. Introduction precipitation and ice melt. Numerous physiological adaptations are apparent in the larvae: heat shock proteins are continuously up- Despite the dominance of insects and other terrestrial arthro- regulated (Rinehart et al., 2006), agents to counter oxidative stress pods throughout the world, only a few species are found in are present in abundance (Lopez-Martinez et al., 2008), loss of Antarctica. Most are collembolans and mites, with the Class Insecta a high percentage of body water is tolerated and in fact contributes represented by only two endemic species of midges (Diptera: to enhanced freeze tolerance (Hayward et al., 2007; Elnitsky et al., Chironomidae) (Convey and Block, 1996). Of these, the range of the 2008). Dramatic changes in metabolites (Michaud et al., 2008) and more abundant midge Belgica antarctica extends further south on gene expression (Lopez-Martinez et al., 2009) accompany these the Antarctic Peninsula than that of any other insect. physiological responses to environmental stress. B. antarctica has a patchy distribution on the Peninsula, but it is In addition to these distinctive biochemical features of cells of particularly abundant near penguin rookeries on the small, off- B. antarctica, it is quite possible that structural features of this shore islands near Palmer Station. In this habitat, midge larvae are insect may also deviate somewhat from the patterns observed in subjected to a range of environmental stresses including desicca- insects at lower latitudes. The harsh environment of Antarctica tion, freezing, anoxia, pH ﬂuctuation from the nitrogenous run-off, obviously places great demands on the resiliency of cells that are and inundation from seawater as well as fresh water from exposed to periodic desiccation and freezing. To promote formation of ice crystals within extracellular spaces rather than within cells, insect cells must rapidly exchange water with their extracellular environments. Expansion of luminal and/or basal * Corresponding author. Tel.: þ1 217 333 6590; fax: þ1 217 244 3499 surface areas of epithelial cells would facilitate this rapid E-mail address: [email protected] (J.B. Nardi). exchange.

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Special emphasis in this manuscript has been placed on the cellular architecture of the three well-delineated epithelial regions of the insect alimentary canal – foregut, midgut, and hindgut. Secretion from the midge’s large salivary gland cells aggregates detritus particles and facilitates uptake of detritus into the gut (Oliver, 1971). After passage of ingested food through the midge larva’s short and narrow foregut, digestion and absorption of ingested material occurs across the peritrophic membrane and microvilli that line the lumen of the midgut. Subsequently digested food passes into the hindgut where ions, small molecules and water are absorbed across a cuticular lining. Comparisons of internal gut morphology of arthropods complement existing studies of external morphological features and molecular characters on which traditional phylogenetic relationships, representing genetic differences among organisms, are based. Although the alimentary canals of other species in the family Chironomidae have not been examined at the cellular level of organization examined in this manuscript, a compre- hensive comparison of internal morphological characters among related species would reveal differences that presumably are based on environmental adaptations. In this study we explore the possibility that the extreme environment in which these midge larvae live shapes the architecture of their gut epithelial cells.

2. Materials and methods

Larvae of B. antarctica were collected from sites near penguin rookeries on Cormorant and Humble Islands, near Palmer Station, Antarctica (64460S, 64040W) in January and February 2007. The high nitrogen run-off from penguin rookeries provides the nutrient base for the microbes, algae (Prasiola crispa), lichens and moss with which midge larvae are usually associated. Larval development is restricted to the brief austral summer, from late December until mid-February; and larvae remain immobilized in the frozen substrate for the remainder of the year. Two years are required for the larva to complete its development (Usher and Edwards, 1984), and adult life is compressed into a 1–2 week period in late December/early January (Sugg et al., 1983). Larvae collected in Antarctica were shipped to the laboratory at Ohio State University where they were maintained with their substrate at 4 C. Digestive tracts and salivary glands from 32 individuals were dissected with fine watchmaker’s forceps in Grace’s insect culture medium. Tissues were immediately fixed at 4 C in a primary fixative of 2.5% glutaraldehyde (v/v) and 0.5% paraformaldehyde (w/v) dissolved in a rinse buffer of 0.1 M cacodylate (pH 7.4) containing 0.18 mM CaCl2 and 0.58 mM sucrose. After 3 h in this fixative, tissues were washed three times with rinse buffer before being transferred to secondary fixative (2% osmium tetroxide dissolved in rinse buffer, w/v) for 4 h. After thoroughly washing with rinse buffer, tissues were gradually dehydrated in a graded ethanol series (10–100%, v/v). From absolute ethanol, tissues were transferred to propylene oxide and infiltrated with mixtures of propylene oxide and resin before being embedded in pure LX112 Fig. 1. (a) Whole mount of larval gut with the three major divisions of the gut resin. demarcated with arrows: fm ¼ foregut–midgut boundary; mh ¼ midgut–hindgut Semithin sections for light microscopy were mounted on glass boundary. The stomodeal valve lies immediately posterior to fm (see Fig. 4). The slides and stained with 0.5% toluidine blue in 1% borax (w/v). sclerotized head capsule is attached at the anterior end and four Malpighian Ultrathin sections were mounted on grids and stained briefly with tubules attach at the midgut–hindgut boundary. (b) Lateral view of the larva. Dorsal is to the right. Beneath the relatively translucent integument of the three saturated aqueous uranyl acetate and Luft’s lead citrate to enhance thoracic segments (arrows point to prothoracic and metathoracic segments), lie contrast. Images were taken with a Hitachi 600 transmission imaginal discs for the three pairs of legs. Dorsal to these leg discs in the meso- electron microscope operating at 75 kV. thoracic and metathoracic segments are wing and haltere discs, respectively. Scale Whole mounts were prepared by fixing tissues at room bar ¼ 1.0 mm. temperature for 30 min in a 4% solution of paraformaldehyde (w/v) dissolved in phosphate-buffered saline (PBS). After Author's personal copy

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Fig. 2. Whole mount of Belgica salivary gland as viewed with Nomarski optics (a) and after labeling DNA with DAPI (b). The salivary duct is located at the arrow. Scale bar ¼ 50 mm.

several rinses in PBS, nuclei of cells were labeled with 1:1000 Malpighian tubules that converge with the alimentary canal at dilution of 40,6-diamidino-2-phenylindole (DAPI, 1 mg/ml the junction of midgut and hindgut (Fig. 1, mh). A prominent water) after ﬁrst permeabilizing cells for 30 min in a solution of stomodeal valve occupies the interface between foregut and 0.1% Triton X-100 in PBS (v/v). Tissues were mounted under midgut (Fig. 1, fm). The foregut occupies only about 5% of the cover glasses in a solution of 70% glycerin in 0.1 M Tris at pH total length of the alimentary canal, while the endodermal 9.0 (v/v). midgut that occupies the region between fm and mh in Fig. 1 clearly represents more than half the length of the alimentary 3. Results canal.

3.1. Global organization of the Belgica larval gut 3.2. Salivary glands and foregut

The larval gut is a straight alimentary canal that is associated Conspicuous salivary glands occupy the anterior end of the with a pair of salivary glands at its anterior end and four larval midge. These glands are both polyploid and polytene (Fig. 2).

Fig. 3. Cuticles of foregut and epidermal epithelia have different stratiﬁcation. At higher magniﬁcation, the internal folded foregut cuticle (a) is compared with the cuticle of the external epidermis (b). A bracket extends across the foregut cuticle. The gut lumen (*) is surrounded by relatively thin cuticle compared to the thicker epidermal cuticle in (b) epithelial cells (e), pigmented fat body (f). In (c) the convoluted integument of the foregut (arrowhead) is surrounded by muscles (arrows), tracheoles (t), neural tissue (n) and salivary gland (g). Scale bars: (a) 1.0 mm, (b) 5 mm, and (c) 20 mm. Author's personal copy

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Like glands found in other larval members of the Chironomidae, intussusception of the foregut epithelium into the midgut (Figs. these salivary glands secrete silken threads that entrap food 4a–d and 5a–c). This folding of the foregut epithelium and its particles. cuticle creates a caecum that is lined centrally by foregut cuticle On the foregut’s apical surface, the cuticle lining the narrow and peripherally by midgut microvilli. The interface of foregut foregut lumen is about 0.3–0.5 mm thick (Fig. 3a). Unlike the thicker, and midgut lies at the anterior end of the caecum. At this contiguous cuticle of the larva’s exoskeleton with its inner electron- junction of foregut and midgut, a peritrophic membrane origi- dense layer (Fig. 3b), this foregut cuticle is highly convoluted and its nates and lines the lumen of the more posterior midgut outermost layer is electron-dense. Conspicuous muscle layers epithelia. surround the foregut. The large salivary glands and larval brain in turn surround these muscles on the basal surface of the larval 3.4. Spatial differentiation of the midgut epithelium foregut (Fig. 3c). An abrupt epithelial discontinuity marked by disparity in 3.3. Stomodeal valve at foregut–midgut interface midgut microvillar length occurs at the interface between the stomodeal region and the more posterior midgut epithelium After being channeled through a foregut lumen lined by (Fig. 4c and 5d). Certain cells at this interface are specialized for a convoluted cuticle, the contents of the gut pass through secretion (Fig. 5d–f) and possibly are endocrine cells. These cells a conspicuous stomodeal valve into the endoperitrophic space of at the posterior edge of the stomodeal valve were the only cells of the midgut epithelium. The stomodeal valve represents an the midgut observed to contain conspicuous secretory granules

Fig. 4. The stomodeal valve represents an intussusception of posterior foregut epithelium (fe) into anterior midgut epithelium (me). (a) The folded foregut epithelium is surrounded by the anterior midgut. Anterior is to the right. (b, c) Longitudinal sections show inner lumen cuticle (ic) and outer lumen cuticle (oc) of the foregut. Between these two cuticles lie two foregut epithelial layers and an enteric muscle layer (m). Midgut epithelium is the outermost layer of the valve. (c) Represents the region delimited by the rectangle in (b). The morphological discontinuity is indicated by the arrow. The peritrophic membrane (p) lies in the lumen separating foregut and midgut. (d) The concentric arrangement of three epithelia, two lumina and one muscle layer is shown in this transverse section of the valve. From periphery to center: (1) midgut epithelium with microvilli lining the lumen in which the peritrophic membrane forms; (2) foregut epithelium faces this lumen and (3) enteric muscles (m) occupy the space between this foregut epithelium and the foregut epithelium facing the innermost lumen. Scale bars: (a, b, d) 50 mm; (c) 20 mm. Author's personal copy

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Fig. 5. At higher resolution, longitudinal sections of the stomodeal valve reveal details of peritrophic membrane formation and the presence of special secretory cells. (a) At the interface between the foregut epithelium and midgut epithelium lies a conﬂuence of muscle (m), foregut epithelium covered by cuticle (fg) and secretory microvilli (arrows) of midgut epithelium. Anterior is at the bottom. (b, c) Copious secretion of peritrophic membrane material (*) occurs from the microvilli of midgut epithelial cells at the anterior end of the stomodeal valve. Note the high density of mitochondria in the adjacent midgut cells. In (b) the newly formed peritrophic membrane (arrow) lies between the foregut (fg) cuticle and the tips of the microvilli. (d) Distinctive secretory cells (arrow) lie within the midgut epithelium of the stomodeal valve at the border between midgut cells with microvilli and midgut cells without obvious microvilli (See Fig. 4c). The gut lumen is at upper left. (e, f) Close-ups of the secretory cell showing the nucleus (n), rough enoplasmic reticulum (arrowhead) and the high density of secretory granules (*). Scale bars: (a) 10 mm; (b, c) 2 mm; (d) 5 mm; (e) 1 mm; and (f) 0.2 mm. Author's personal copy

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(Fig. 5e and f). Posterior to the stomodeal valve, a striking the base of microvilli on the luminal surfaces of these midgut gradient of microvillar length occurs along the antero-posterior cells (Fig. 8c) offer an obvious route for the cellular uptake of (AP) axis of the midgut epithelium (Fig. 6), with short (w0.1 to these electron-dense particles from the ectoperitrophic space of 0.2 mm) microvilli occupying anterior regions of the midgut and the gut lumen. extremely long, straight and densely packed (w10 mm, Figs. 7 and Infoldings of the basal membranes of epithelial cells are most 9) microvilli occupying posterior regions of the midgut. This prominent in the anterior and the posterior regions of the midgut morphological gradient is evident in longitudinal sections of the (Figs. 7c, 8e, and 9c); mitochondria are most conspicuous along the midgut (Fig. 6a–c) as well as the series of transverse sections from basal surface of the anterior midgut (Fig. 7a, c); and infoldings of different locations along the AP axis of the Belgica gut (Figs. 6d–f the basal plasma membrane contain numerous mitochondria and and 7–9). electron-lucent vacuoles (Figs. 7c and 9c). Underlying the antero-posterior topography revealed by the Regenerative cells are scattered throughout the larval midgut microvilli of the midgut is a parallel topography, at the base of the epithelia and presumably represent imaginal stem cells that microvilli, reflected by contours of the apical surfaces of the midgut replace the larval epithelium at metamorphosis. These cells are cells. These surfaces are most convoluted in regions of the midgut located basally in the larval epithelium and are densely packed with the shortest microvilli and are least convoluted in regions of with ribosomes and endoplasmic reticulum (Fig. 10). the midgut with the longest microvilli (Fig. 6d–f). Rough endoplasmic reticulum (RER) is present throughout all 3.5. Contents of the midgut lumen regions of the midgut (Figs. 7d, 8d, and 9d). Stacks of large flattened sacs of endoplasmic reticulum are especially evident in 3.5.1. Endoperitrophic space the posterior region of the midgut epithelium. Some smooth Within the gut lumen, the peritrophic membrane separates an endoplasmic reticulum (SER) is interspersed among the RER of inner endoperitrophic space from an outer ectoperitrophic space the anterior third of the midgut, with little if any SER is observed (Terra and Ferreira, 1994; Fig. 6). The anterior endoperitrophic in the middle third or the posterior third of the midgut. However, space of the midgut is packed with relatively intact multicellular clearly defined Golgi complexes are not evident in any of the microbial organisms. Gradual digestion of microbes is reflected in midgut cells. the disappearance of pigmentation from the gut lumen in the The lumen of the middle third of the Belgica midgut is lined posterior third of the midgut (Fig. 1a). Within the confines of the with microvilli of intermediate length (w1 mm) that are associ- midgut’s peritrophic membrane, microbial cells arranged singly or ated with electron-dense particles of uniform size (w0.05 mm). in various aggregates can be readily discerned. These cells can be These particles lie within the ectoperitrophic space and show identified as predominantly nonbacterial microbes – i.e., algae, a strong affinity for the microvilli (Fig. 8a–c). Within the cyto- fungi, lichens, protists (Fig. 11a–c). plasm of the underlying midgut cells, electron-dense particles of identical size are concentrated in autophagic vacuoles, each 3.5.2. Ectoperitrophic space delimited by a plasma membrane, and are presumed to enter The surrounding ectoperitrophic space is lined by midgut these epithelial cells by endocytosis. Numerous coated vesicles at epithelium with microvilli whose lengths are graded along the

Fig. 6. Longitudinal (a–c) and transverse (d–f) sections of midgut epithelium posterior to the stomodeal valve show regional differences along the antero-posterior axis of the gut. Three equally spaced regions along this axis are illustrated: (a, d) anterior region; (b, e) middle region; (c, f) posterior region. In each image the microvillar surface and gut lumen are at top. The peritrophic membrane (arrow) is visible in (a, b, d, and e). Scale bars ¼ 20 mm. Author's personal copy

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Fig. 7. These are ultrastructural images of the anterior region of the midgut epithelium. Note the high concentration of mitochondria along the basal surface of the epithelium in (a) as well as the dark cell (arrow) at the basal surface of this epithelium. Lumen is marked with an asterisk (*). (b) The epithelial surface facing the lumen (*) is covered by short microvilli. (c) Mitochondria (arrows) are localized among membrane infoldings that lie adjacent to the basal lamina and enteric muscles (arrowhead). (d) Perinuclear region of midgut cell. Rough endoplasmic reticulum (ER) marked with arrowhead. Smooth ER marked with double arrowhead. Arrows point to mitochondria. n ¼ nucleus. Scale bars: (a) 5 mm; (b, c) 2 mm; and (d) 1 mm.

antero-posterior axis. Within one of the 10 whole mounts of hindgut cells appears in whole mounts as a zone of cells with small larval guts prepared, gregarines with distinctive appendages nuclei among the polyploid nuclei in cells of the larval hindgut were found in this midgut zone (between the peritrophic matrix epithelium. and midgut epithelial surface) (Fig. 11d and e). Considering the Immediately posterior to the imaginal ring of epithelial cells, isolation of these gregarines from other related host species, the anterior third of the hindgut is lined by a smooth cuticle these protists most likely represent a distinct species of dipteran approximately 0.25 mm thick. This hindgut cuticle, like the parasite. foregut cuticle, is contiguous with the exoskeleton. Also, like the foregut cuticle, the hindgut cuticle secretes a well-delineated, 3.6. Spatial differentiation of the hindgut electron-dense outermost cuticular layer. The arrangement of these different layers secreted by hindgut epithelial cells is 3.6.1. Ring of undifferentiated, presumptive adult distinct from the arrangement of the cuticular layers secreted by hindgut epithelium epidermal epithelia (Fig. 3b). The anterior hindgut cuticle is In the images of the Belgica gut labeled with DAPI (Fig. 12a), secreted by attenuated, highly folded extensions of the apical a discontinuity in nuclear labeling is observed at the midgut– surfaces of the hindgut epithelium containing conspicuous hindgut boundary. At the anterior-most region of the larval mitochondria and separated by large vacuoles that lack electron- hindgut, an imaginal ring of undifferentiated presumptive adult density (Fig. 12b and c). Author's personal copy

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Fig. 8. Different magniﬁcations of the middle region of the midgut are illustrated in (a–e). In images a and c, the midgut lumen is at the top; in e, it is down. Electron-dense particles occupy the space between the peritrophic membrane (arrow in a) and the microvillar surface. Comparable particles are observed in autophagic vacuoles of midgut cells (arrowheads in b). (c) Higher resolution images suggest that these particles are taken up by endocytosis (arrows) at the microvillar surface. (d) Perinuclear region of midgut cell. Arrowheads indicate rough endoplasmic reticulum; arrows point to mitochondria; n ¼ nucleus; me ¼ membrane between two cells. (e) Basal lamina (arrowhead) and muscles (m) cover the basal surface of this epithelium. Infoldings of the basal surface of plasma membrane are not conspicuous. Scale bars: (a) 5 mm; (b, c) 1.0 mm; (d) 1 mm; and (e) 2 mm.

While apical ends of epithelial cells in the anterior third of the this posterior-most hindgut epithelium serves a mechanical hindgut have conspicuous large vacuoles, the central region of the function. In this most posterior portion of the hindgut, the lumen hindgut is occupied by epithelial cells that lack vacuoles but that is devoid of both resident microbes as well as ingested microbes have extremely convoluted apical membranes characteristic of (Fig. 12f). epithelial cells involved in active transport of ions and water (Fig. 12d and e). Also in the central region of the hindgut, the 3.7. Muscles associated with the gut epithelia presence of the electron-dense bacteria observed in high-resolution images is reﬂected in the pigmentation of the central portion of The basal surface of the hindgut epithelium, like the foregut the hindgut as viewed in whole mounts of larval guts (Fig. 1a). epithelium, is closely apposed to a uniformly thick layer of circum- In the posterior third of the hindgut or rectum, the highly ferential muscles (w10 mm) (Figs. 3c, 12e, f). By contrast, muscles convoluted cuticle lining the lumen mirrors the structure of the associated with the midgut epithelium are sparsely but regularly foregut. The apical surfaces of the epithelial cells of the rectum distributed over the midgut’s basal surface (Figs. 6–9). Relatively lack infolded membranes associated with mitochondria and are widely dispersed muscle cells lie on the basal surface of the midgut apparently not specialized for transport of ions and water. The epithelium and are embedded in the matrix of the epithelial basal basal surface of this region, by contrast, is highly infolded. lamina. These represent the longitudinal muscles of the gut. Sparsely Association of this basal surface with numerous muscles suggests distributed circumferential muscles are also present. Author's personal copy

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Fig. 9. In a–c, the midgut lumen is to the left. Different magniﬁcations of the posterior region of the midgut are illustrated. (a) Note numerous clear vacuoles (arrows) that lie between the luminal microvilli and the basal lamina (lower right). (b) The long microvilli are densely packed and extend approximately 10 mm into the lumen. (c) The basal membranes of cells in this posterior region are highly folded. Basal lamina is indicated with arrowheads. Muscle ¼ m. (d) Perinuclear region of midgut cell. Arrowheads indicate rough endoplasmic reticulum; arrows point to mitochondria. Scale bars: (a) 10 mm; (b) 5 mm; (c) 2 mm; and (d) 1 mm.

4. Discussion how general or how unique internal features are among insects, however, awaits additional structural studies on other related The study of insect diversity and evolution has been insect species. advanced by extensive surveys of molecular phylogeny and morphology of external integuments; however, knowledge and 4.1. Differentiation of foregut–midgut boundary: structure appreciation of insect diversity remain incomplete without of the peritrophic membrane and stomodeal valve adequate knowledge of the diversity of internal anatomy/physiology of insects and how this diversity is inﬂuenced by A specialized luminal region at the foregut–midgut boundary environment. can be visualized even in whole mounts of the alimentary canal With the paucity of information on the cellular architecture of (Fig. 1a). In cross-sections and longitudinal sections of the insect guts, however, associating particular gut epithelial struc- alimentary canal at this boundary region, an inner concentric tures with adaptation to particular diets and/or environments ring of folded foregut epithelium and associated muscle layers remains in a rudimentary state. Conventional descriptions of gut lies within the anterior midgut epithelium (Figs. 4, 5). Among epithelial diversity (Lehane, 1998; Noble-Nesbitt, 1998; Terra the Diptera, the degree of specialization of foregut and midgut et al., 1988) clearly do not consider the marked regional differ- epithelial cells varies among the suborders. The most complex entiation of microvilli on midgut cells of B. antarctica to be specialization of the foregut–midgut interface is found among a common feature of epithelial cells of insect guts. Establishing the muscoid ﬂies, in which specialized anterior midgut Author's personal copy

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epithelium is closely apposed to the stomodeal valve to form the distinctive cardia (Eisemann et al., 2001; Binnington, 1988); but the simplest specialization of the foregut–midgut interface is observed in the suborder Nematocera, of which Belgica is a member. In these ﬂies, the foregut has been described as forming a short intussusception into the anterior midgut referred to as the stomodeal valve (King, 1991; Wigglesworth, 1930). For Belgica, however, this intussusception of foregut as a percentage of total foregut surrounded by midgut is higher than that reported by Volf et al. (2004) for four other nem- atoceran Diptera in the families Culicidae (Culex pipiens) and Psychodidae (Lutzomyia longipalpis, Phlebotomus duboscqi, Phle- botomus papatasi). Peritrophic structures for many insect species have often been described as chitinous, reticulated membranes with chitin micro- ﬁbrils in a hexagonal or orthogonal arrangement (Lehane, 1998). These peritrophic structures consist of chitin networks embedded in protein–carbohydrate matrices (Wang and Granados, 2001; Tellam et al., 1999). The origin and consistency of peritrophic membranes, gels and matrices differ among the insects (Terra, 2001; Binnington et al., 1998). The peritrophic structures of some insects, such as lepidopteran larvae, have traditionally been described as arising from cells along the length of the midgut epithelium (type I peritrophic matrix). Recent studies involving labeling of lepidopteran peritrophic proteins have indicated that while one peritrophic protein (i.e., invertebrate intestinal mucin) is secreted by epithelial cells throughout the length of the midgut (Harper and Granados, 1999), certain peritrophic proteins recognized by an antibody raised against the peritrophic membrane of Heliothis virescens are produced by specialized cells near the foregut–midgut interface (Ryerse et al., 1992). At the junction of foregut and midgut epithelia in Diptera, the peritrophic structure (type II) arises from microvilli of midgut epithelia (Eisemann et al., 2001) and lines the lumen of the more posterior midgut epithelia. In Belgica, the midgut epithelial cells of the stomodeal valve are the only cells observed to produce a copious secretion associated with a newly formed structure that represents a type II peritrophic membrane.

4.2. Regional differentiation of midgut epithelium

At least in some insects, the midgut is differentiated both structurally and functionally along its length (Lehane,1998; Marana et al., 1997; Ferreira et al., 1990; Terra et al., 1988; Dow, 1981). The marked and graded differences in microvillar lengths observed for Belgica midgut epithelial cells, however, represent an extreme example of such regional differentiation (Fig. 6). Extensive infolding of the basal epithelial surface of midgut and hindgut cells, however, as frequently observed in other insects, is only evident in certain regions (anterior third and posterior third) of the Belgica midgut and the posterior third (rectum) of its hindgut (Villaro et al., 1999; Lehane, 1998; Marana et al., 1997). See Figs. 7c, 8e, 9c and 12f. The high concentrations of electron-dense particles that occupy the ectoperitrophic space of the central region of the midgut are not observed elsewhere in the alimentary canal. The presence of comparable particles in autophagic vacuoles of these midgut cells suggests that these particles are taken up by cells rather than secreted by gut cells. The high concentration of particles in the gut lumen also implies that their movement proceeds toward the low Fig. 10. Regenerative cells (asterisks) of midgut epithelium are scattered throughout concentration of particles observed within the midgut epithelial the larval epithelium. Cells from different regions along the antero-posterior axis are cells. shown. (a) Anterior third. (b) Middle third. (c) Posterior third. Arrowheads point to Although regenerative cells have not been observed in basal laminae; m ¼ muscles; n ¼ nuclei of regenerative cells. Scale bars ¼ 2.0 mm. midgut epithelia of certain immature arthropods such as larval Author's personal copy

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Fig. 11. The contents of the midgut lumen are described in both the endoperitrophic space (a–c) and the ectoperitrophic space (d, e). The multicellular microbial forms observed in these thin sections (a, b, c) of the midgut’s endoperitrophic space are fungal and algal. (d, e). An unidentiﬁed but distinctive gregarine protist inhabits the ectoperitrophic space. Note the spiral appendages (arrows) visible on these gregarines. p ¼ peritrophic membrane; m ¼ midgut epithelium; en ¼ endoperitrophic space. Scale bars: (a, b) 20 mm; (c) 3 mm; (d) 50 mm; and (e) 20 mm.

Musca domestica (Terra et al., 1988) and immature Allacma fusca The rectal pads and/or papillae of many terrestrial insects (Rost-Roszkowska et al., 2007a), regenerative cells (presumptive have been shown to be involved in uptake of water and ions. imaginal stem cells) are observed scattered throughout the Rectal epithelial cells (posterior hindgut) of these terrestrial monolayer of the Belgica midgut epithelium as they are in the insects have conspicuous folding of their apical plasma gut epithelia of many other insects (Rost-Roszkowska et al., membranes. However, the ilea or anterior hindguts of aquatic 2007a,b; Rost, 2006; Ohlstein and Spradling, 2005; Evangelista insects such as Cenocorixa and Ephydrella as well as some and Leite, 2003; Lehane, 1998; Hecker, 1977). In addition to terrestrial insects such as Formica, are adapted for ion and/or high-resolution images of Belgica regenerative cells (Fig. 10), the water transport (Villaro et al., 1999; Marshall and Wright, 1974; dark cell on the basal surface of the anterior midgut epithelium Jarial and Scudder, 1970). Insects living in aquatic or semi-aquatic in Fig. 7a, probably also represents such a regenerative cell. environments presumably have different absorptive activities, being more concerned with absorption of inorganic ions rather than with water absorption. 4.3. Differentiation of hindgut epithelium Based on examination of other insect hindguts (Noble-Nesbitt, 1998; Jarial and Scudder, 1970), numerous vacuoles are correlated A discontinuity in nuclear size at the midgut–hindgut inter- with an absorptive function for these epithelial cells. The anterior face is evident in whole mounts of Belgica guts (Fig. 12a). A ring two-thirds of the Belgica hindgut or ileum seems to be adapted for of epithelial cells with nuclei that are smaller than those nuclei absorption of ions and water in the larva’s semi-aquatic environ- located both anterior and posterior to the ring presumably ment (Murakami and Shiotsuki, 2001; Heuser et al., 1993). Unlike represents the stem cells or presumptive imaginal cells for the the rectal pads or papillae of terrestrial insects, the posterior third adult hindgut (Takashima et al., 2008; Murakami and Shiotsuki, of the hindgut or rectum epithelium of Belgica shows no special 2001). adaptations on its apical surface for transport of ions and water; Author's personal copy

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Fig. 12. Images of the hindgut epithelium are presented as DAPI-labeled whole mounts (a) and thin sections (b–f). Fig. 10a shows the midgut (mg)–hindgut interface of the larval gut that has been labeled with the speciﬁc DNA stain DAPI. At its anterior-most region, the nuclei of the hindgut epithelial cells are dwarfed by the polyploid nuclei of the Malpighian tubules (arrows) and more posterior hindgut cells (*). (b) The anterior third of the hindgut consists of cells with polyploid nuclei (n) whose apical ends are occupied by numerous vesicles and covered by a thin cuticle (arrow). A thin basal lamina (bl) is located on its basal surface. (c) Convoluted membranes, electron-lucent vesicles and mitochondria are concentrated at apical surfaces of this anterior third of the hindgut epithelium. The cuticle is indicated with an arrow. (d, e) Bacteria inhabit the lumen of the middle third of the hindgut. The apical ends of these epithelial cells are covered by a thin cuticle and have extremely convoluted membranes (arrows). Basally the epithelium has a thick muscle layer (m). (f) The cuticle lining the lumen of the posterior third of the hindgut is convoluted (arrow). The basal lamina of epithelial cells has conspicuous infoldings (arrowheads); m ¼ muscles. Scale bars: (a) 50 mm; (b) 2 mm; (c) 1.0 mm; (d) 2 mm; (e) 20 mm; and (f) 2 mm. Author's personal copy

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