Midgut of the diplopod Urostreptus atrobrunneus: structure, function, and redefinition of hepatic cells.

Diplopods are considered important macroarthropods the soil as part of its maintenance and balance. These animals usually do not occur in high densities, but population explosions caused by environmental disturbances, climate changes, and use of pesticides that eliminate possible competitors, have been reported. The millipede Urostreptus atrobrunneus Pierozzi and Fontanetti, 2006 have become a nuisance to humans in infestation sites in urban centers of the state of Sao Paulo, Brazil. As a contribution to the understanding of this potential pest, this study describes the histology, histochemistry, and ultrastructure of the U. atrobrunneus midgut, and presents the redefinition of hepatic cells somewhat controversial in the literature. The region of the midgut is characterized by the absence of a cuticular intima, and composed of a pseudostratified epithelium on a thick basal membrane, followed by a muscle layer, a layer of hepatic cells, lined by an external membrane. The morphology observed in U. atrobrunneus is similar to that reported for other species of diplopods. The hepatic cells have been previously described as randomly without forming a layer, however, the present results clearly demonstrate that these cells form a continuous layer over the whole midgut.

The digestive tract of millipedes is a simple straight tube that starts in the mouth and ends in the anus, and is divided in foregut, midgut, and hingut.Except for the midgut, the entire digestive tube is internally lined by a cuticular intima.Salivary glands are found along the foregut and are responsible for the production of secretions that lubricate the ingested food and contain digestive enzymes that aid in the early stages of digestion (Nunez and Crawford, 1977;Fontanetti and Camargo-Mathias, 1997;Fantazzini et al., 1998;Moreira-de-Sousa and Fontanetti, 2012).
The midgut of millipedes located after the foregut and before the pyloric region, has a fundamental role in the digestive process of the animal.Some enzymes are secreted by epithelial cells on the food particles and others may be produced by microorganisms in the lumen (Hopkin and Read, 1992).
The structure and role of the midgut of diplopods have been described for few species (Bowen, 1968;Nunez and Crawford, 1977;Hubert, 1979bHubert, , 1988;;Fontanetti and Camargo-Mathias, 1997;Fontanetti et al., 2001;Fantazzini et al., 2002;Camargo-Mathias et al., 2004;Sosinka et al., 2014).Studies on Brazilian species are rare and most of the literature is restricted to some orders.Fontanetti and Camargo-Mathias (1997) described the morphology and histology of the digestive tract of the Plusioporus setiger species (Spirostreptida).Fantazzini et al. (2002) described the anatomy of the midgut of Rhinocricus padbergi (Spirobolida) by means of histological and histochemical analysis, and Camargo-Mathias et al. (2004) later characterized the ultrastructure of the midgut of this species.
This study was aimed at describing the morphology of the midgut of U. atrobrunneus, through histological, histochemical, and ultrastructural analysis, and its relationship with the roles played by this organ.It was also aimed to review the terminology and definition of hepatic cells, which are controversial in the literature.

Materials and Methods
The specimens (n=8) were collected by Edilberto Gianotti and by Raphael Baston de Souza in the surroundings of the city of Rio Claro, São Paulo, Brazil (22°23'59"S; 47°34'18"W).The animals were maintained in a terrarium with soil and leaf litter moistened regularly.
The midgut was obtained by dissecting specimens anesthetized with sulfuric ether on a dissection plate with saline solution.The digestive tube was fixed with Bouin solution, 4% paraformaldehyde or formol calcium for 24 hours.The material was embedded in historesin and stained with hematoxylin and eosin for histological analysis.The histochemical stains used were: Xylidine Ponceau (Melo and Vidal, 1980) and Bromophenol Blue (Pearse, 1985) for the detection of proteins; Periodic acid-Schiff (PAS) and simultaneous PAS and Alcian Blue (Junqueira and Junqueira, 1983) for polysaccharides; von Kossa method (Junqueira and Junqueira, 1983) for calcium detection; and Nile Blue (Lison, 1960) and Sudan Black B (Junqueira and Junqueira, 1983) for lipids detection.
The material processed for transmission electron microscopy was fixed with 2.5% glutaraldehyde in 0.1M cacodylate buffer at 4 °C, rinsed with cacodylate buffer and post-fixed with 1% osmium tetroxide for 2 hours.After this period, the material was then rinsed with the same buffer, immersed in 10% ethanol for 15 minutes and contrasted with 2% uranyl acetate in 10% ethanol for 4 hours.Following this procedure, the material was dehydrated in a crescent series of acetone and immersed in resin: acetone (1:1) for 12 hours, embedded in Epon-araldite resin with catalyzer for 24 hours and placed in the oven at 70 °C for 24 hours for resin polymerization.Sections were obtained with ultramicrotome and observed with a Phillips CM 100 transmission electron microscope.

Histology
The midgut of U. atrobrunneus consists of a pseudostratified epithelium resting on a basal lamina, followed by a muscle layer, a layer of hepatic cells, and irregularly distributed muscles, lined externally by a membrane termed external membrane.Adjacent to the midgut there is the perivisceral fat body (Figure 1A).
The epithelium is composed of three cell types: principal or absorptive cells, generative or regenerative cells and secretory cells.The absorptive cells are the most numerous cells, which exhibit a well-defined brush border.The generative cells are smaller and located at the base of the epithelium (Figure 1B).The secretory cells are among the absorptive cells and release a secretion of the apocrine type, in which part of the cell is lost along with the secretion (Figure 1B).
Just beneath the muscle layer are the hepatic cells forming a continuous layer.These cells are irregular and have a spherical nuclei and heterogeneous cytoplasm (Figure 1A, B).
Along the entire midgut, a peritrophic membrane consisted of acellular layers is observed in the lumen (Figure 1A, B).

Histochemistry
The histochemical analysis revealed that the epithelium was strongly positive to proteins, while hepatic cells were moderately to weakly stained (Figure 2A, B).
The secretion released by epithelial cells contains glycoproteins.Some vesicles were strongly stained for proteins (Figure 2A, B) while others were more intensely stained for neutral polysaccharides.
Neutral polysaccharides were observed in the brush border, in the basal membrane, and some hepatic cells (Figure 2C).
Calcium was observed diffusedly in the absorptive cells (Figure 2D), and small quantities of lipids were present in the brush border of epithelial cells and some hepatic cells (data not shown).

Ultrastructure
Absorptive (principal) cells exhibit many microvilli in their free face (Figure 3A) and rest on a thick basal lamina, where cytoplasmic projections are observed (Figure 3B).The cytoplasm of absorptive cells exhibits well-developed rough endoplasmic reticulum (Figure 3C, D) and spherocrystals (Figures 3A, D, 4A).
It is possible to observe that the materials in spherocrystals accumulate in concentric layers; spherocrystals are enclosed by a membrane and are in close contact with the rough endoplasmic reticulum (Figure 3D).Spherocrystals found in the cytoplasm of absorptive cells are easily removed during the preparation for microtomy, resulting in empty sites (Figure 4A, B).
Among the absorptive cells observed, secretory cells were seen releasing part of their cytoplasm with secretion (Figure 3A).
The generative cells, located at the base of the epithelium, exhibit large nucleus, an evident nucleolus, cytoplasm less electron-dense than that observed in absorptive cells with several mitochondria more electron-dense than that observed in absorptive cells (Figure 4C).The contact of epithelial cells occurs through interdigitations and occlusive junctions.However, in many regions, dilated intercellular spaces are generally observed (Figure 4D).
Hepatic cells exhibit heterogeneous cytoplasm (Figure 4A), large quantities of mitochondria, vacuoles of various aspects and large nucleus (Figure 4A, B).Many empty spaces are observed in the cytoplasm of these cells, due to the preparation for microtomy that removed granules present in the cells (arrows in Figure 4A, B).Between hepatic cells, hemocytes in different maturation stages are observed (Figure 4B).

Discussion
The midgut of U. atrobrunneus exhibited a pseudostratified epithelium.Bowen (1968) has reported the presence of a pseudostratified columnar epithelium in the midgut of Floridobolus penneri and Narceus gordanus, both Spirobolida.For all other species of diplopods examined, a simple prismatic epithelium has been reported.This has been confirmed by Hubert (1979b) on the ultrastructural analysis of Cylindroiulus londinensis.On the contrary, in R. padbergi, the ultrastructural analysis has revealed a pseudostratified epithelium (Camargo-Mathias et al., 2004).Sosinka et al. (2014) reported that the midgut epithelium of the species P. lagurus, A. gigas and J. scandinavius is columnar pseudostratified.In this study, was also observed that the epithelium of the midgut of U. atrobrunneus is pseudostratified.
The absorptive cells, also called by some authors as principal cells or digestive cells, are involved in both the absorption of food an in the transport elements to the hemolymph.There are descriptions that these cells accumulate heavy metals such as lead, cadmium, zinc and copper (Köhler at al., 1995) and calcium (Hubert, 1988;Fantazzini et al., 2002).
The observation of absorptive cells in diplopods, including U. atrobrunneus demonstrated the presence of numerous secretory vesicles.These vesicles are in the lumen near the microvilli, attesting that the absorptive cell can play the role of secretory cell according to the physiological demand.The content of these vesicles varied in coloration, suggesting a maturation level or a dehydration process of the secretion during the transport from the cytoplasm to the intestinal lumen, as also proposed by Fantazzini et al. (2002) and Fontanetti et al. (2001).
Similar characteristics have been described for the digestive cells mite.According to Filimonova (2008), these cells are polyfunctional, once that digestion processes, absorption and excretion are carried out by cells in the different phases of the cell cycle.The author observed the presence of various different cell types representing a gradual change from the absorptive cells for secretory cells (Filimonova, 2008).The same should happen in the midgut of millipedes.
In diplopods, the mode of secretion can be apocrine, as observed in the present study and described by Fontanetti et al. (2001) for the species P. setiger, by Fantazzini et al. (2002) for R. padbergi, or merocrine as observed by Hefner (1929) for Parajulus impressus.The latter author also suggested that in other millipedes the type of secretion may be holocrine.Sosinka et al. (2014) reported the release of microapocrine secretion in the millipedes Julus scandinavius and Archispirostreptus gigas.The vesicle are formed at the end of microvilli and then discharged into the lumen as double-membrane vesicles.
The generative or regenerative cells are located in the basal area of the epithelium and are distributed along the entire midgut.These cells are responsible for epithelial renewal (Hopkin and Read, 1992) and are considered unipotent stem cells of millipedes midgut (Sosinka et al., 2014).Hubert (1988) reported that hepatic cells are not contiguous, and that the cohesion between them is ensured by a connective tissue.Regarding its embryonic origin there are no studies, however Hubert (1988) states that the hepatic cells are lined by a coelomic leaflet, which may indicate that they are of mesodermal origin.
On the similarity between these cells with those that form the fat body of these animals, many authors have confused the hepatic cells with the fat body, a totally separate body (Subramoniam, 1972;Fontanetti and Camargo-Mathias, 1997;Fantazzini et al., 1998;2002).Further, according Seifert and Rosenberg (1977), the hepatic cells are distributed randomly, not forming a layer, while the results shown here indicate that these cells in fact form a continuous layer throughout the entire midgut.The hepatic cells of millipedes probably perform functions similar to the hepatopancreas of other arthropods or chloragogeneous tissue surrounding the gut of the earthworms (Hopkin and Read, 1992).According to Hubert (1978b) these are cells rich in glycogen and probably interfere in intermediary metabolism.
According Hubert (1988) the hepatic cells branch out towards the basal portion of the midgut cells.According to the author the fusiform junctions suggest that there is a transport between the two cell types.In the present study, observed that hepatic cells form a continuous layer along the entire midgut, but without connections with epithelial cells.
Part of the products of digestion assimilated by the absorptive cells will pass to the hepatic cells (Hopkin and Read, 1992).Hubert (1978aHubert ( , b, 1988) ) reports that the intestinal cells and hepatic cells perform complementary functions.The intestinal cells are involved in the absorption of the digestion products, while hepatic cells are involved in mineral retention.The hepatic cells can also accumulate toxic substances which are transported by hemolymph and then excreted.The hepatic cells actively participates in detoxification process of the organism.(Kohler, 2002;Nogarol andFontanetti, 2010, 2011;Perez and Fontanetti, 2011;Merlini et al., 2012).These observations explain the fact that large quantities of spherocrystals are found in the cytoplasm of these cells in millipede U. atrobrunneus.
The histochemical analysis has revealed that the epithelium and hepatic cells were positive for proteins, indicating a high concentration of proteins in the region.Except for secretory cells, the epithelium is weakly stained for neutral polysaccharides, suggesting that this epithelium participates in the production of neutral polysaccharides, but do not store them.Lipid staining techniques indicated the presence of this compound in small quantities in the brush border in hepatic cells, indicating that the midgut of U. atrobrunneus has little participation in the metabolism of these elements.
The presence of calcium in the midgut of diplopods is well known, since these animals dwell in the soil.The presence of this element represents a form of detoxification, a well-described process for invertebrates, including diplopods (Fontanetti et al., 2006).
According to Fantazzini et al. (2002), the presence of calcium in absorptive cells suggests that the epithelium might be involved in the transportation of this element.In this case, the metal precipitation occurs as intracellular granules of different types which, after being stored, are directly discharged by secretory vesicles into the lumen or by substitution of the intestinal epithelium (Hubert, 1979a;Nogarol and Fontanetti, 2010;Godoy and Fontanetti, 2010).
Hopkin and Read (1992) also observed several granules of calcium phosphate arranged concentrically in intestinal cells of diplopods.According to Hubert (1979b), since the midgut is a site of mineral storage, it plays an important role in the ionic regulation of the organism, and consequently, the cyclic discharge of granules may be a form of excretion.The excretion of minerals in U. atrobrunneus may occur in a similar way, since several spherocrystals has also been observed in the midgut of this species and the accumulation of material inside them is also concentric.
According to Fontanetti et al. (2006), spherocrystals may be named mineralized bodies, and their structural organization can vary inside different cells, from different parts of the body, with distinct aspects.These authors have also observed that spherical bodies were enclosed by a membrane, although not associated to organelles such as the endoplasmic reticulum or the Golgi apparatus.In U. atrobrunneus, spherocrystals were also enclosed by a membrane, but in close contact with the rough endoplasmic reticulum.
According Camargo-Mathias et al. (2004), the structural organization of the basal portion of the absorptive cells in the midgut of R.padbergi supports the hypothesis that in addition to absorbing compounds from food, these cells may also be involved in the transport of elements from the hemolymph.For these authors, the morphology is similar to that of renal tubular cells of vertebrates and play a role in the transport of ions, the large number of mitochondria in this region suggests a similar role for absorptive cells (Camargo-Mathias et al., 2004).Analyzing the results of the present study, the same may occurs with U. atrobrunneus, since a significant quantity of mitochondria was also observed.
It concludes that the midgut structure millipede U. atrobrunneus is very similar to that described for other species.In the species studied in this work the epithelium is pseudostratified.The observation of hepatic cells in this species contributed to clarify a point of controversy in the literature sometimes these cells were referred to as "fat body layer midgut" and sometimes described as randomly arranged cells without forming a layer.It is clear from this analysis that these cells form a continuous layer around the entire midgut.