Ultrastructural and functional analysis of secretory goblet cells in the midgut of the lepidopteran Anticarsia gemmatalis

Abstract

Defoliation caused by Anticarsia gemmatalis larvae affects the commercial production of the soybean. Although regulation of the digestion of soybean components has become part of the suggested strategy to overcome problems caused by Anticarsia larvae, few studies have focused on the morphological and cellular aspects of Anticarsia intestinal tissue. We have therefore further analyzed the morphology and ultrastructure of the midgut of 5th instar larvae of A. gemmatalis. Dissected midgut was subjected to chemical or cryo-fixation and then to several descriptive and analytical techniques associated with both light and electron microscopy in order to correlate anatomical and physiological aspects of this organ. Histological analysis revealed typical anatomy composed of a cell layer limited by a peritrophic membrane. The identified lepidoptera-specific goblet cells were shown to contain several mitochondria inside microvilli of the goblet cell cavity and a vacuolar H+-ATPase possibly coupled to a K+-pumping system. Columnar cells were present and exhibited microvilli dispersed along the apical region that also presented secretory characteristics. We additionally found evidence for the secretion of polyphosphate (PolyP) into the midgut, a result corroborating previous reports suggesting an excretion route from the goblet cell cavity toward the luminal space. Thus, our results suggest that the Anticarsia midgut not only possesses several typical lepidopteran features but also presents some unique aspects such as the presence of a tubular network and PolyP-containing apocrine secretions, plus an apparent route for the release of cellular debris by the goblet cells.

Introduction

In the past few decades, the food and agricultural industry has shown a growing dependence on the consumption of soybean. Because of this increasing demand, the cultivation and precurement of soybean have recently shown tremendous improvements. Nevertheless, the industry has suffered an important impact because of the presence of the lepidopteran Anticarsia gemmatalis Hubner (Lepidoptera: Noctuidea), which is still one of the most prevalent pests of the soy crop. Despite the importance of this pest for the world’s agricultural industry, the amount of information on the biology of Anticarsia is relatively scant. For example, only a few studies have reported the cell biology and physiological aspects of the digestive system of Anticarsia (Gomes et al. 2012; Levy et al. 2004) and most have focused on the impact of pest management strategies (Levy et al. 2007; Matos et al. 1999; Sousa et al. 2010). This becomes more surprising as the ultrastructure of the digestive system of other lepidopteran models has been extensively studied (Anderson and Harvey 1966; Cioffi 1979, 1984; Pinheiro et al. 2008).

As in other insects, the lepidopteran gut consists in three main regions: foregut, midgut and hindgut, among which the midgut is the best-studied region. The morphology of the midgut has been better described in the Bombycoidea superfamily after the seminal descriptions in Manduca sexta L. (Cioffi 1979) and Hyalophora cecropia L. (Anderson and Harvey 1966). These studies helped to establish the principles of the cellular and structural organization of a lepidopteran midgut, which include a basement lamina formed by acellular components, trachae and muscle cells, on the basal side and a peritrophic membrane (peritrophic matrix), on the luminal side, limiting a single layer of epithelial cells. This cell layer is composed of columnar and regenerative cells, also present in other insects and by goblet cells (GC), which are a typical cell type of Lepidoptera but which are not homologous to the vertebrate GC or to any other insect cell so far described.

The importance of an ultrastructural approach to the understanding of a systemic function has been demonstrated in various insect models. In M. sexta, early ultrastructural descriptions of the midgut showed the existence of GCs characterized by a microvilli-rich extracellular cavity (GC) that invaginates toward the cell cytoplasm. Two distinct features of the microvilli of GCs draw attention to: (1) the abundant presence of mitochondria in the cytoplasmic portion of the microvilli along the anterior and middle midgut, suggesting an intense energy demand within this region (Anderson and Harvey 1966; Cioffi 1979) and (2) a granular electron-dense pattern at the ultrastructural level (Harvey et al. 1983). This granular pattern (the portasome) is probably the result of a vast accumulation of transmembrane proteins driving an ATP-dependent K+ gradient inside the GC by a “K+-pump system” (Dow et al. 1984; Harvey et al. 1983). The K+-pump is now known to be composed of two independent counterparts: a vacuolar H+-ATPase (V-ATPase) responsible for the establishment of an H+ gradient (Schweikl et al. 1989; Wieczorek et al. 1991) and a 2:1 K+/H+ exchanger (Azuma et al. 1995; Grinstein and Wieczorek 1994). This K+ gradient energizes the uptake of amino acids by specific transporters (Giordana et al. 1998; Hanozet et al. 1980; Hennigan et al. 1993a, 1993b). The K+/H+ exchanger seems to be a specific feature of Lepidoptera taxa and its identity is yet to be determined. On the other hand, stimulation of nutrient uptake by the presence of a V-H+-ATPase has subsequently been shown to be a common pattern found in other insect models (Harvey et al. 2010), a characteristic that is not shared by vertebrates.

In view of the importance of the knowledge of cell biology for an understanding of physiological processes and for the design of possible alternative pest control strategies, the present work reports a detailed description of the ultrastructure of 5th instar larvae of A. gemmatalis based on a variety of microscopical techniques. The overall structure of the Anticarsia midgut is similar to those from other closely related lepidopterans, although several features of interest are described herein. Briefly, electron microscopic images suggest a route for the elimination of degenerated organelles at the GC cavity. We have used specific antibodies to confirm the presence of the V-ATPase pump at the microvilli of GCs, a characteristic that is related to the presence of a potassium gradient detected by X-ray microanalysis of high-pressure frozen samples. X-ray microanalysis has also revealed a new set of calcium-rich vesicles in the columnar cells. Apocrine secretions have been observed as polyphosphate (PolyP)-rich vesicles budding from the columnar cells.

Materials and methods

Reagents

Antibodies against the subunit e and E of the M. sexta V-ATPase were kindly provided by Prof. Helmut Wieczorek and Dr. Markus Huss, University of Osnabrück, Germany. 4′,6-diamidino-2-phenylindole (DAPI) was purchased from Sigma–Aldrich, antibody against phosphorylated histone H3 (pHH3) from Santa Cruz, Alexa-Fluor-488-conjugated anti-mouse antibody from Invitrogen and Historesin from Leica Microsystem. Glutaraldehyde, paraformaldehyde, sodium cacodylate and osmium tetroxide were purchased from Electron Microscopy Sciences. All other chemicals and reagents were of analytical grade.

Insect colonies

Anticarsia gemmatalis was reared in a laboratory colony kept at 27°C and 70% relative humidity as described (Gomes et al. 2012). Adults were maintained in plastic cages and paper sheets were provided for egg deposition. After 24 h, eggs were transferred to a plastic box until hatched and until larvae development. Larval development was followed visually and 5th instar larvae were obtained within 10 or 11 days after hatching.

Chemical fixation for morphological analysis

Fifth instar larvae were dissected and the gut of each was isolated, briefly washed in phosphate-buffered saline (PBS: 137 mM NaCl, 2.7 mM KCl, 10 mM phosphate, pH 7.4) at room temperature and fixed with 4% formaldehyde, 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer, pH 7.2, at room temperature for 2 h. Fixed guts were washed in 0.1 M sodium cacodylate buffer and further prepared according to the protocols described below.

For light microscopy (LM), the gut was dehydrated in an ethanol-graded series and embedded into progressive Historesin concentrations. After embedding of the midgut, Historesin hardener was added and the samples were incubated at 60°C for 24 h. Semi-thin sections were prepared, adhered to glass slides and stained with a drop of Giemsa followed by washes with ethanol. Slides were observed with a Zeiss Axioplan microscope equipped with a digital camera (Olympus XC-30).

For transmission electron microscopy (TEM), the gut was post-fixed in 1% OsO4, 0.8% FeCNK, 5 mM CaCl2 for 1 h in the dark at room temperature. Samples were washed and dehydrated by an ethanol-graded series, followed by embedding into progressive Epon concentrations. Epon-embedded samples were hardened at 60°C for 72 h and 80-nm ultrathin sections were prepared and attached to copper grids. Lead citrate and uranyl acetate were used for post-staining and samples were observed with a transmission electron microscope (JEOL 1200 EX equipped with an Olympus Megaview III digital camera). Alternatively, 0.1 M phosphate buffer pH 7.0 was used instead of sodium cacodylate for tissue fixation. In this case, post-fixation was performed in 1% OsO4.

For scanning electron microscopy (SEM), the gut was dehydrated with ethanol and critical-point dried. Samples were manually fractured with a razor blade to expose the cytoplasmic surface and gold-sputtered, after which they were observed under a FEI Quanta 250 scanning electron microscope operating at high vacuum (below 10−2 Pa). Alternatively, the gut lumen was partially exposed after fixation, adhered to a stub with silver glue and observed with a FEI Quanta 250 scanning electron microscope operating at a variable pressure of 80–120 Pa.

High-pressure freezing and freeze-substitution

Midguts were dissected and small fragments were rapidly transferred to a high-pressure holder containing 1-hexadecene. The samples were frozen in a Bal-Tec HPM 010. Freeze substitution was performed by using 1.45% KF, 3% glutaraldehyde, 1% OsO4 in methanol (Hardt and Plattner 2000). Samples were kept at −80°C for 72 h, −20°C for 6 h and 4°C for 4 h, washed with acetone and embedded into progressive concentrations of Epon.

Immunohistochemistry and detection of PolyP stores

PolyP stores were detected in cryosections as previously described (Gomes et al. 2012). Briefly, samples were fixed and incubated overnight in 10% and 30 % sucrose. Each gut was then immersed in Tissue-TEK and frozen by immersion in LN2. Semi-thin sections of 8–10 μm were obtained in a cryostat and allowed to thaw at room temperature for 30 min. Sections were washed in PBS, incubated in 0.1 μg/ml DAPI and mounted with n-propyl-gallate. Slides were observed in a Zeiss Axioplan microscope equipped with a XC-30 Olympus digital camera and a filter set optimized for PolyP imaging by DAPI (excitation: 350 nm, emission above 500 nm). After image acquisition, deconvolution was performed by using a no-neighborhood algorithm that is available in Olympus Cell^P software and that attenuates out of focus light by removing low-frequency data at the Fourier space (Wallace et al. 2001). Alternatively, actin imaging was performed by incubation of cryosections in 1:150 phalloidin-fluorescein-isothiocyanate (FITC) in 2% bovine serum albumin, 0.3% Triton X-100, PBS (blocking buffer) for 2 h. The sections were washed in blocking buffer and then in PBS and mounted with n-propyl-gallate for observation in a Zeiss Axioplan microscope equipped with a XC-30 Olympus digital camera via a FITC filter set.

For immunohistochemistry of pHH3 or V-ATPase e, larvae were dissected at the indicated time point and the gut of each was briefly washed in PBS and fixed in 4% formaldehyde, 2.5% glutaraldehyde in 0.1 M sodium cacodylate pH 7.2 buffer for 2 h. After fixation, the gut was washed in 0.1 M sodium cacodylate buffer and incubated in blocking buffer (see above) for 2 h, followed by incubation in 1:50 anti-pHH3 or 1:100 anti V-ATPase for 2 h in blocking buffer. After being washed in blocking buffer, the gut was incubated in 1:500 anti-mouse Alexa-488-conjugated secondary antibody for 2 h, washed and mounted in n-propyl gallate. Negative controls were performed by omission of primary antibodies and incubation with blocking buffer only. Images were taken by using a Zeiss Axioplan microscope equipped with a XC-30 Olympus digital camera or a Leica confocal microscope system.

When expressed, the relative intensity of pHH3 staining along the gut axial length was measured after removal of the red channel (containing most of the gut autofluorescence) by using ITEM software.

Results

General morphology

The gut of each 5th instar larvae of A. gemmatalis was dissected under a stereomicroscope and prepared for various microscopical analysis. Images showed that the gut comprises a reduced foregut with no apparent appendices, a long tubular midgut apparently lacking local specializations and a short hindgut connected to the Malpighian tubules that extended along the medial length of the midgut to which they were attached (Fig. 1a). A continuous layer of circular muscular tissue supported the midgut along its whole extension (Fig. 1b), whereas longitudinal muscles seemed more discontinuous and apparently robuster. Larval gut remained full except during ecdysis or upon starvation. The granular gut contents remained separated from the epithelium by a peritrophic membrane (Fig. 1c, f) and could only be readily visualized when minimal handling was performed, as the fixation and dehydration steps seemed to wash away a large fraction of the gut contents (not shown). The epithelial layer was fringed (Fig. 1d, e) and seemed to secrete a viscous material (Korayem et al. 2004; Wang and Granados 1997) that was removed during sample preparation. This viscous secretion surrounded the densely packed microvilli (Fig. 1e) leaving the aperture of the GC cavity exposed (Fig. 1e, inset). Analysis of various sections showed that the morphological organization of the midgut was basically limited by a monolayer of columnar cells and GCs that presented a stochastic distribution (Fig. 1f). GCs were more easily identified by the presence of an extracellular cavity that remained partially connected to the lumen from a specific position along the axial length (Fig. 1f, asterisks). The peritrophic membrane was well preserved (Fig. 1g), allowing a clear delimitation of the endoperitrophic and ectoperitrophic spaces (Fig. 1f). A fragmented bolus was only observed in the endoperitrophic region, whereas the ectoperitrophic space was characterized by the presence of apocrine secretions of various densities, sometimes seen in close contact with the peritrophic membrane (Fig. 1f, g). Secretion-rich cytoplasmic regions inside the columnar cells were observed, although the cellular origin of the apocrine secretions was not determined.

Fig. 1
figure1

General morphology of the midgut of Anticarsia gemmatalis. The general morphology was evaluated by means of several microscopic techniques. a General organization of freshly dissected Anticarsia gut observed under a stereomicroscope (fg foregut, mg midgut, mt Malpighian tubules, h hindgut). b Variable-pressure scanning electron microscopy (VPSEM) of a hydrated gut showing the malpighian tubules (arrows). c VPSEM after exposure by tissue rupture showing a food bolus (fb) inside the endoperitrophic space. d Conventional SEM of the critical-point-dried epithelial surface at low magnification. e Higher magnification of the cells in d showing the microvilli (mv) and extracellular secretion products (arrows) that became visible after removal of mucous material during sample preparation. Inset The apical apertures (arrows) of the goblet cells were also observed by VPSEM. f Semi-thin section of the cellular layer and peritrophic membrane (pm) limiting the endoperitrophic space (ens) showing the presence of products of apocrine secretion (arrows) on the ectoperitrophic space (ecs). Note also the columnar cells (c), goblet cells (g), goblet cell cavities (asterisks) and basal lamina (BL). g Transmission electron microscopy (TEM) image showing the peritrophic membrane (pm) surrounding the food bolus (fb) and lying in close proximity to apocrine secretions (as). Bars 5 mm (a), 200 μm (b), 300 μm (c), 500 μm (d), 20 μm (e), 100 μm (e, inset), 100 μm (f), 2 μm (g)

Using an antibody against a pHH3, which has been described as a marker of mitotic fate (Grosse et al. 2011; Ribalta et al. 2004), no labeling was observed when the gut from 5th instar animals were used (not shown). On the other hand, a banding pattern was observed when larvae at ecdysis from the 4th to 5th instar were used (Supplemental Fig. 1a). This suggested that most of the cellular renewal and tissue growth occurred during ecdysis. Accordingly, we observed that cell positioning at the basal lamina, namely the site at which regenerative cells are located (Supplemental Fig. 1b, inset), also changed during ecdysis (Supplemental Fig. 1b, c), i.e., cells lying side by side and at higher numbers were frequently seen at later times as if they had just undergone cell division (Supplemental Fig. 1c). Nevertheless, we did not observe any mitotic patterns such as nuclei division, duplication of centrioles, chromosome migration, or cytokinesis, suggesting that cell renewal might take place at an early stage during ecdysis.

Goblet cell

The most remarkable feature of a GC is a cavity termed the GC cavity (Figs. 1f, 2a-c). SEM images showed that the surface of the GC presented a dense array of microvilli (Fig. 2a). Fluorescence microscopy of cells incubated with phalloidin-FITC revealed that these cavities were surrounded by actin (Fig. 2b). Microvilli were concentrated mainly at the basal portion of the GC where they also seemed to be longer than their apical GC microvilli counterparts (Fig. 2c). This was observed in several images in several different orientations, both by light (Fig. 1f) and electron (Fig. 2c) microscopy. A basal labyrinth supported the basal region of the GC providing an increased surface area at the basal epithelium (Fig. 2d). Mitochondria were seen inside the GC microvilli (Fig. 2e) suggesting intense energy consumption in this region. The GC environment was separated from the gut luminal space by the valve-like packing of microvilli containing no mitochondria (Fig. 2f).

Fig. 2
figure2

Goblet cell and goblet cell cavity. a SEM of fractured midgut showing the exposed surface of a goblet cell cavity (asterisk). b Phalloidin-fluorescein-isothiocyanate staining revealed by fluorescence microscopy around the goblet cell cavities (asterisk). c, d TEM images detailing the goblet cell cavity (gc) and basal labyrinth (blb), respectively, after chemical fixation (n nucleus). e, f TEM images showing the presence of mitochondria within the microvilli (arrows) and the goblet cell valve (v) at the goblet cell aperture, respectively (gc goblet cell, RER rough endoplasmic reticulum, mv microvilli). Bars 30 μm (a), 40 μm (b), 15 μm (c), 1 μm (d), 300 nm (e), 1 μm (f)

In M. sexta, the GC has been demonstrated to contain a V-H+-ATPase that energizes the transport of K+ to the GC cavity, maintaining an ionic balance spatially isolated from the lumen. This is usually characterized by a compact granular structure on the plasma membrane related to the high density of transmembrane proteins termed portasomes (Cioffi and Harvey 1981; Harvey et al. 1981). Antibodies against the 9.3-kDa e subunit of V-H+-ATPase of M. sexta (Merzendorfer et al. 1999) were localized to the GC microvilli in the midgut of A. gemmatalis (Fig. 3a, b; Supplemental Movie 1) thus supporting the assumption that the K+-pump system is also present in Anticarsia. Similar results were obtained by using antibodies against the subunit E of the M. sexta V-H+- ATPase (data not shown). The typical granular profile of portasomes could not be observed at the membrane level, either by chemical or cryo-fixation. This might have been the result of the lower density of the V-H+-ATPase in this cell type as suggested by immunocytochemistry with LR-White-embedded material in which only a few gold particles were observed at the microvilli membranes (Fig. 3b, inset). The use of K+ as an ionic agent has been suggested as a specific feature in the Lepidoptera, as other insect models have been shown to employ Na+ to couple amino acid uptake (Harvey et al. 2010; Meleshkevitch et al. 2009; Rheault et al. 2007). Elemental mapping by X-ray microanalysis supported this K+ hypothesis as an extremely low level of sodium was detected along the midgut extension, whereas potassium seemed to be present at much more substantial levels (Fig. 3c). Phosphorous levels were used as a control of homogeneous acquisition conditions along the whole midgut. Accordingly, X-ray microanalysis of GC cavities revealed detectable levels of potassium (Fig. 3d).

Fig. 3
figure3

Presence of a vacuolar ATPase at the goblet cell microvilli. Immunolocalization of V-H+-ATPase in the midgut of A. gemmatalis in whole tissue (a) or in a transversal section of OCT-embedded material (b) showing labeling of the goblet cell cavity (arrows). Immunogold labeling demonstrated that the pump was mainly localized to the microvilli (b, inset). Electron probe X-ray mapping of the midgut (c) by SEM revealed the distribution of phosphorous (d), potassium (e) and sodium (f) along the gut (se secondary electron image, mg midgut). g Electron probe X-ray microanalysis of a goblet cell cavity from a section prepared by high-pressure freezing and KF freeze substitution. Bars 300 μm (a), 200 μm (b), 200 nm (b, inset)

Columnar cell and secretion routes

Together with GCs, columnar cells are the most prevalent cell types along the midgut of A. gemmatalis larvae. Densely packed microvilli dispersed along the apical region and in direct contact with the ectoperitrophic space were observed by TEM or LM in cross-section profiles (Fig. 4a). Vesicles apparently lay within a secretion route concentrated along the apical region of the columnar cells (Fig. 4a, b) but were also found in a basal position, although in a smaller number (data not shown). Mitochondrial profiles were concentrated in a layer below the apical region (Fig. 4c). An extensive tubular network was observed in the same region (Fig. 4d). Multivesicular bodies were less commonly found (Fig. 4e). Compared with GCs, columnar cells presented a larger cytoplasmic volume. Rough endoplasmic reticulum extended from the perinuclear region (Fig. 4f) toward the microvilli region (Fig. 4g) apparently occupying a large cytoplasmic area (Fig. 4h). When potassium fluoride was used to precipitate calcium in situ (Hardt and Plattner 2000) during freeze substitution of high-pressure frozen tissue, an extensive array of calcium-rich electron-dense compartments were seen including cytoplasmic bodies originating from the Golgi complex (Fig. 4i) as confirmed by X-ray microanalysis (Supplementary Fig. 2)

Fig. 4
figure4

Columnar cells. a–d General morphology of columnar and goblet cells (gc goblet cell cavity) observed by TEM detailing the presence of secretory vesicles (arrows in a, b) close to the microvillar layer (mv), mitochondria (asterisks in c) and a tubular network (t). e Multivesicular bodies (asterisks) in the cytoplasm of columnar cells were occasionally observed (m mitochondrion). f Endoplasmic reticulum profiles (er) in the perinuclear region (n nucleus). g Rough endoplasmic reticulum (rer) close to microvilli (mv). h Rough endoplasmic reticulum (rer) adjacent to lateral membranes. i KF freeze substitution of high-pressure frozen sections revealing a diversity of calcium stores (arrow) near a Golgi complex (g). Bars 10 μm (a), 2 μm (b, g), 1 μm (c, d, f, h, i), 500 nm (e)

Secretion vesicles were a prominent feature of columnar cells. Three distinct patterns of apocrine vesicles were observed: (1) vesicles carrying a large portion of amorphous cytoplasmic content (Fig. 5a), (2) vesicles also containing internal membrane profiles (Fig. 5b), or (3) degenerating subcellular structures resembling autophagic bodies (Fig. 5c). Some apocrine vesicles were shown to contain considerable amounts of inorganic polyphosphate (PolyP), as observed in fluorescence microscopy images of DAPI-stained sections (Fig. 5d). The presence of unstained regions within these vesicles suggested that PolyP was restricted to the regions surrounding the inclusions seen in Fig. 5c. Myelin figures, suggestive of an autophagic process, were found in the cytoplasm of columnar cells (Fig. 5e) and seemed to aggregate at the median-apical region below the microvilli (Fig. 5f). Microapocrine vesicles were also found around the microvilli layer of columnar cells (Fig. 5g). Interestingly, the goblet microvilli also seemed to be a site of residual secretion of degraded components (Fig. 5h, i).

Fig. 5
figure5

Secretion and autophagy in the midgut of A. gemmatalis. a–c TEM images of apocrine vesicles containing amorphous cytoplasmic content (a), fibrilar material (b) and autophagic bodies (c). d Localization of PolyP stores (arrows) in OCT-embedded samples by DAPI staining (inset higher magnification view). e, f TEM images of autophagic bodies and their distribution (arrows in f) along a columnar cell. g TEM image of microapocrine vesicles (arrows) budding from the columnar cell microvilli. h, i TEM images showing the release of residual cytoplasmic material into the goblet cell cavity. Bars 4 μm (a, c), 10 μm (b), 20 μm (d), 2 μm (e, f), 1 μm (g), 500 nm (h, i)

Basal lamina

Epithelial cells were observed sitting on a basal lamina oxygenated by tracheal tubes connecting the midgut to the tegument, which ramified into several tracheoles (Fig. 6a, arrows). Tracheoles were limited to the extracellular space but penetrated relatively deeply into the cellular region by basal invaginations (Fig. 6b). Circular muscle cells supporting the gut remained in the basal lamina (Figs. 1b, 6c) and were usually visualized in cells submitted to chemical fixation as an almost empty space (Fig. 6c; Cioffi 1979, 1984). Nevertheless, high-pressure freezing followed by freeze substitution revealed a more complex native structure in which a compact layer of amorphous material devoid of empty spaces filled the basal lamina (Fig. 6d, e).

Fig. 6
figure6

Basal lamina of A. gemmatalis midgut. a VPSEM of the tracheolar system (arrows) in connection with the midgut (mg). b TEM image of the invagination of the tracheal system (arrows) via basal infoldings of the epithelium (bl basal lamina, blb basal labyrinth, gc goblet cell cavity, N nucleus). c TEM image of the basal lamina (bl), circular muscle cells (m) and cellular layer (cl). d, e TEM images of high-pressure frozen samples showing the basal lamina (bl), basally located muscles cells (M, m) and cellular layer (cl). Bars 500 nm (a), 2 μm (b), 5 μm (c–e)

Discussion

A. gemmatalis is one of the most common pests of soybean plantations. Nevertheless, most information concerning the biology of this organism is inferred from studies with other lepidopteran species. A fine characterization of A. gemmatalis organs and tissues is, therefore, fundamental for the proper comprehension of the physiological and structural aspects of this organism. In this regard, our results have shown that the overall morphological organization of A. gemmatalis midgut agrees with the general model of Lepidoptera (Anderson and Harvey 1966; Baldwin and Hakim 1991; Cioffi 1979, 1984; Pinheiro et al. 2008; Sousa et al. 2010), although some specific characteristics can be attributed exclusively to A. gemmatalis.

As found in other Lepidoptera (Anderson and Harvey 1966; Cioffi 1979, 1984; Pinheiro et al. 2008), the general midgut organization of A. gemmatalis comprises a cellular monolayer composed of three cell types: columnar, goblet and regenerative cells. Although the presence of endocrine cells has been suggested in other insect models, their characterization has not been examined here because of the lack of a consensus for the overall picture of their morphological characteristics. As has also been pointed out, endocrine cells might have been erroneously identified as regenerative cells (Dow 1986). At the basal lamina, we have observed a muscular tissue that supports the gut and that is oxygenated by a tracheal system connecting the gut to the tegument and the external environment. On the luminal side, a peritrophic membrane surrounds the midgut and establishes an endo- and ectoperitrophic space. The observation of accumulated food particles limited to the endoperitrophic space reinforces previous descriptions that digestion initially occurs in the endoperitrophic space and progresses toward the intermediate and final steps on reaching the ectoperitrophic space and the microvillar layer (Santos et al. 1983; Schumaker et al. 1993; Terra et al. 1979; Terra and Ferreira 1994).

The observation of a partial isolation of the GC cavity from the luminal space by a valve-like structure is important on the basis that this is the site at which the formation of a K+ gradient used to drive nutrient uptake takes place (Harvey and Nedergaard 1964; Wolfersberger et al. 1982; Wood et al. 1969). The presence of an isolation port, presumably formed by specialized microvilli, might be essential to the mechanisms involved in midgut digestion, which is known to use K+ as a driving force, another characteristic feature of the Lepidoptera; in Diptera, Na+ is used instead (Harvey et al. 2009; Okech et al. 2008; Rheault et al. 2007). At present, we have not measured whether Anticarsia GCs create a K+ gradient but its structural similarity to other lepidopterans and the K+ X-ray elemental profiles obtained seem to suggest that this is indeed the case. The microvillar area that forms a dense zone was readily recognized by light and electron microscopy and showed the presence of mitochondria inside microvilli suggesting intense energy demands in this region. In other models, this has been attributed to the intense ATP demand by a V-ATPase that plays a role in the formation of an H+-gradient (Schweikl et al. 1989), which is thus used by an 2:1 K+/H+ exchanger to establish a K+ gradient (Azuma et al. 1995). Genes of the V-ATPase complex have been cloned in M. sexta (Dow 1992; Graf et al. 1992, 1994a, 1994b; Merzendorfer et al. 1999, 2000; Wieczorek, et al. 1999) and have homologs in both human and yeast genomes. In Western blot assays, antibodies against the subunit e of the V-ATPase recognize a single peptide of 10 kDa in extracts of Anticarsia midgut (data not shown) and co-localize in the microvilli of Anticarsia GC (Fig. 3), as has been shown in M. sexta (Reineke et al. 2002; Vitavska et al. 2003). The molecular identity of the K+/H+ exchanger, however, remains to be identified. In Diptera, an analogous function is performed by a Na+/H+ exchanger, like that cloned from Anopheles gambiae Giles (agNHA1; Rheault et al. 2007). However, the use of an antibody against agNHA1 did not label specific features of the Anticarsia midgut and a continuous labeling of the apical microvilli in the midgut was seen (data not shown).

In M. sexta and in Erinnyis ello, the morphology of the GC cavity of the anterior and posterior midgut changes; the GCs of the posterior midgut occupy smaller cell volumes and possess fewer mitochondria inside their microvilli (Cioffi 1979). These differences have been related to a role in water secretion and absorption (Santos et al. 1984). We have not observed such a clear distinction. This might be attributable to the lack of anatomical evidence for the division of the midgut into sub-regions. Future work should enable the determination of molecular markers to evaluate degrees of segmentation along the Anticarsia midgut. Nevertheless, apart from investigations into the creation of a K+ gradient along the midgut, few studies concerning the GC have been undertaken. The observation of an apparent excretion of cellular components at the microvilli of the GC cavity has corroborated previous work showing that spherites of A. gemmatalis follow a secretion route at this site (Gomes et al. 2012). This combined information suggests that a physiological connection between the GC cavity and mechanisms of cellular detoxification exists. The region might serve as an intermediate storage unit or delivery space, before the removal of cellular debris, to the luminal region. Whether this corresponds to a possible "two-step excretion" mechanism is intriguing. Further studies should evaluate whether similar mechanisms are found among other lepidopterans.

In addition to their functional role in the recycling of cellular components, enzymes produced by the midgut epithelia must be packed and sent to the lumen to take part in digestion. In most insects, this is accomplished both by the formation of microapocrine vesicles (Espinoza-Fuentes and Terra 1987; Santos et al. 1986) and by exocytosis via secretory vesicles (Berner et al. 1983; Bignell et al. 1982; Lehane 1976; Moffatt and Lehane 1990). Accordingly, we have observed both features along the Anticarsia midgut, being concentrated at the columnar cells. The presence of extensive areas of rough endoplasmic reticulum further supports the functional role of this cell type in the continued synthesis and secretion of digestive enzymes. We have also observed a tubular network in the medial-apical cytoplasm of the columnar cell. The biological significance of these observations remains unclear and similar observations in other lepidoterans have not yet been reported. The endoplasmic reticulum and tubular network might mediate the transport of enzymes and membranes to the microvillar and luminal region or represent an intermediate step toward the production of apocrine vesicles.

Apocrine secretion has also been observed adjacent to or budding from columnar cells, despite the presence of secretory granules and microapocrine vesicles in this region. Apocrine secretions are characterized by the release of portions of the apical cytoplasm and are a common feature of the insect gut (Terra and Ferreira 1994). Interestingly, a detailed examination of apocrine vesicles by TEM has revealed the presence of internal structures resembling residual autophagic compartments. Similar features have not been extensively described in other models, although they have been found in at least one other invertebrate (Rost-Roszkowska et al. 2012). Future studies need to evaluate the presence of autophagic markers at this subcellular site in order to link morphological and physiological data. As apocrine vesicles might be important for cell renovation processes that take place in the insect midgut (Humbert 1979; Terra and Ferreira 1994), a connection between the clearance of autophagic bodies and cell renewal might exist in the insect midgut. The finding of regenerative cells at the basal side (not extending toward the luminal region) suggests that the division of regenerative cells is the starting point for a differentiation process occurring during the molt (as suggested by midgut labeling with pHH3) at a discrete position of the midgut, as in other models. The packing of undifferentiated cells during the molt as observed by TEM is similar to that described for M. sexta (Baldwin and Hakim 1991) and is reminiscent of the overall aspect of stage D of differentiation, when cells are elongating and extending toward the midgut lumen. The lack of mitotic figures at this stage seems to corroborate this observation, implying that cell division occurs early during the molt. Of interest will be an evaluation of whether cell differentiation and growth occur rapidly after the onset of molting period, or whether they occur at the beginning of the intermolt period. The massive increase of size observed in the Anticarsia body during intermolt stages suggest that cell growth largely takes places after molting, as in other Lepidoptera.

The localization of PolyP by using DAPI as a PolyP probe (Gomes et al. 2008; Ramos et al. 2010; Tijssen et al. 1982) has shown a preferred localization of this polymer in apocrine vesicles in the midgut of A. gemmatalis larvae. The presence of PolyP in such structures suggests that this polymer is released into the extracellular space, as occurs during the degranulation of human platelet (Ruiz et al. 2004). In a previous study, the presence of PolyP bodies in the midgut of A. gemmatalis has been described (Gomes et al. 2012), together with its release to the extracellular space from the GC. Thus, the accumulation and release of PolyP is another common feature of columnar and GCs and is, as far as we know, the only report of PolyP release to the extracellular space in invertebrate models. The biological significance of the PolyP storage in apocrine vesicles is not understood but its compartmentalization together with autophagic bodies is curious. Heparin (a polyanion with similar biochemical properties to PolyP) is known to play a role in the blood coagulation of vertebrates by binding with antithrombin (a plasma serpin that acts as a serine protease inhibitor; Jin et al. 1997; Thunberg et al. 1982). Interestingly, PolyP has also been shown to bind to at least one serpin and to one vertebrate serine protease in vitro (Church et al. 1988). Other classes of serpins have also been shown to inhibit cysteine and aspartic proteases (Kamada et al. 1997; Liu et al. 2003; Mathialagan and Hansen 1996; Silverman et al. 2004; Takeda et al. 1995) and to regulate cell death processes (Bird et al. 1998; Medema et al. 2001; Suminami et al. 2000; Tewari et al. 1995). We have previously described a role for PolyP in the regulation of an aspartic-like protease in the eggs of Rhodnius prolixus Stahl (Gomes et al. 2010). The role of PolyP regulation in protease-triggered events such as digestion and cell death in the midgut of Anticarsia is currently under investigation in our laboratory.

Finally, most descriptions of the organization of the insect midgut relies on the observation of chemically fixed tissues, which have usually been dehydrated by organic solvents such as ethanol and acetone and are thereby susceptible to preparation artifacts. In this report, the use of microscopic techniques that do not rely on the chemical fixation of the gut has allowed us to observe a series of features of the lepidopteran gut that have not previously been seen in chemically fixed cells. As an example, SEM analysis of hydrated guts by using variable-pressure SEM, has enabled the observation of an extracellular mucinous layer covering the microvilli of the epithelial cells. We hypothesize that this accounts for a second site of protection and isolation of the epithelial membrane against microorganism invasion or abrasion by food particles, as also demonstrated for the peritrophic membrane. In addition, the basal lamina has been usually described, at the ultrastructural level, mostly as an empty region (Anderson and Harvey 1966). Here, high-pressure frozen and freeze-substituted samples have revealed the basal lamina as a more complex region filled with an amorphous material. Another interesting approach will be to evaluate to what extent the development of these routines for immunocytochemistry will allow the localization of the extracellular matrix components to provide the basis for an understanding of the scaffold of such structures in insects. Finally, the heterogeneity of calcium-rich compartments found in the columnar cells deserves further attention. In the blowfly Calliphora vicina, calcium stores are important for the activation of V-ATPases by a serotonin-cAMP pathway (Dames et al. 2006; Rein et al. 2006; Zimmermann 1997). Similarly, when starved or during ecdysis, V-ATPases of the midgut of M. sexta are downregulated (Reineke et al. 2002). The calcium stores detected in the midgut cells of A. gemmatalis might have similar physiological and functional roles.

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Acknowledgements

We express our gratitude to Prof. Helmut Wieczorek and Dr. Markus Huss, University of Osnabrück for providing antibodies of the subunit e M. sexta and to Dr. Eduardo Fox and Prof. Wanderley de Souza for proofreading.

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Correspondence to E. A. Machado or K. Miranda.

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This work was supported by grants from the following Brazilian agencies: Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq, INCT de Entomologia Molecular and INCT de Biologia Estrutural e Bioimagem), Coordenação de Aperfeiçoamento do Pessoal de Nível Superior (CAPES), Fundação Carlos Chagas Filho de Amparo à Pesquisa de Estado do Rio de Janeiro (FAPERJ) and CAPES-Petrobrás.

The funders of this work had no role in the study design, data collection and analysis, decision to publish, or preparation of this article.

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Supplemental figure 1
figure7

a Whole tissue immunohistochemistry with anti-pHH3 of the gut of Anticarsia larvae under ecdysis showing a banding pattern at discrete regions of the gut. The black bars above mark the regions stained by the antibodies. The graph below represents the relative intensity of the staining along the gut axial length. b TEM image showing the organization of cells at the basal lamina during the 5th instar. Inset position of the regenerative cells (asterisk) close to the basal lamina (bl, BL) and below a goblet cell (gc) cavity. c TEM image showing cells during ecdysis from the 4th to 5th instar. Note that they present a densely packed organization (gc goblet cell). Bar: B – 5 μm, B, inset – 15 μm, C – 5 μm. (JPEG 40 kb)

Supplemental figure 2
figure8

Typical electron probe X-ray microanalysis of the vesicles depicted in Fig. 2 prepared from high-pressure frozen tissues, freeze-substituted in the presence of KF as a calcium precipitating agent. (JPEG 5 kb)

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High resolution image (TIFF 1292 kb)

High resolution image (TIFF 66 kb)

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Gomes, F.M., Carvalho, D.B., Machado, E.A. et al. Ultrastructural and functional analysis of secretory goblet cells in the midgut of the lepidopteran Anticarsia gemmatalis . Cell Tissue Res 352, 313–326 (2013). https://doi.org/10.1007/s00441-013-1563-4

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Keywords

  • Goblet cell
  • Polyphosphate
  • Ultrastructure
  • Midgut
  • Lepidoptera
  • Anticarsia gemmatalis