Histology of the Ileum in Bees (Hymenoptera, Apoidea)
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REGULAR PAPER HISTOLOGY OF THE ILEUM IN BEES (HYMENOPTERA, APOIDEA) Carolina Gonçalves Santos and José Eduardo Serrão Department of General Biology, Federal University of Viçosa (UFV), Viçosa, MG, Brazil. ABSTRACT In most hymenopterans, the ileum extends as a long tube from the midgut to the rectum, and shows no marked anatomical specialization. The function of the ileum is not fully understood, although the presence of an epithelial layer of cuboidal or columnar cells with apical and basal plasma membrane infoldings suggests that this organ may be involved in water and nutrient absorption. In this work, we investigated the ileal morphology of 47 species of bees in the Andrenidae, Apidae (Apini, Meliponini, Xylocopinae, Centridini, Bombini, Eucerini, Euglossini, Tapinotaspidini, Exomalopsini), Halictidae and Megachilidae, as part of a study to understand the physiological and phylogenetic importance of this organ. In all cases, the ileum consisted of an epithelium containing cuboidal or columnar cells that usually had basal nuclei and apical plasma membrane infoldings, although there were variations in cell shape, position of the nucleus, degree of chromatin condensation and cuticle thickness. The epithelial cells were covered with a cuticle and transverse sections revealed the presence of 4-6 folds projecting into the lumen. The cell cytoplasm below the apical plasma membrane infoldings contained numerous vacuoles of different sizes. A single layer of circular muscle was located beneath the epithelium. The histological organization of the ileum suggested a role in the absorption of luminal solutes as a mechanism for regulating the hemolymph and the general osmotic balance of these insects. However, there was no relationship between the structural organization of the ileum and the degree of social development and/or phylogeny of the bees. Key words: Digestive tract, morphology, osmotic control, physiology INTRODUCTION its length. Although its precise function remains to be The digestive tract of insects is divided into established, the ileum may be involved in water and foregut, midgut and hindgut. The foregut and the nutrient absorption [8]. In the stingless bee Melipona hindgut are of ectodermal origin whereas the midgut quadrifasciata anthidiodes, the epithelial cells of the is endodermic [6]. In bees, the foregut consists of ileum have four types of microvilli, indicating that the pharynx, esophagus, crop and proventriculus, the there may be some form of compartmentalization midgut corresponds to the ventriculus and the hindgut in the absorption of organic solutes; this finding, is divided into ileum and rectum [3,4,10,22,34,39]. together with other ultrastructural features, is also Various morphological and biochemical aspects of suggestive of a digestive function [9,10]. the digestive tract of bees have been studied [7,13,15- Morphological variations in the digestive tract 17,23,25,29,32,41]. However, the ileum had received of insects have been related to differences in their less attention because of the general belief that the feeding habits and degree of sociality [1,4,11,12,18]. midgut is responsible for food digestion and nutrient However, phylogenetic relationships may also absorption whereas the rectum is responsible for influence insect gut morphology [29,32,33,42]. In this osmotic control [32,35-37]. context, studies designed to compare the histological The ileum of bees is a long tube interposed between structure of the ileum in different species of bees are the midgut and the rectum but shows no visible required to test the hypothesis that variations in this (macroscopic) anatomical specialization throughout organ are related to phylogeny and/or the degree of _______________ sociality in these insects. The purpose of this study Correspondence to: Dr. José Eduardo Serrão was to compare the histology of the ileum in bees Departamento de Biologia Geral, Universidade Federal de Viçosa (UFV), belonging to several tribes of four families in order CEP: 36570000, Viçosa, MG, Brazil. Tel: (55) (31) 3899-1301, Fax: (55) (31) 3899-2549. E-mail: [email protected] to assess the foregoing relationships. Braz. J. morphol. Sci. (2006) 23(3-4), 405-413 406 C. G. Santos and J. E. Serrão MATERIAL AND METHODS 2B). The single layered epithelium consisted of only Forty-seven species of bees from the Andrenidae, Apidae one cell type that varied from cuboidal to columnar. (Apini, Meliponini, Xylocopinae, Centridini, Bombini, All of the specimens examined had flattened cells Eucerini, Euglossini, Tapinotaspidini, Exomalopsini), at the ileal-rectal interface (Fig. 1C), with the Halictidae and Megachilidae were studied (Table 1). The ileal epithelial cells usually having apical plasma bees were collected while foraging on flowers in the towns membrane infoldings and basal nuclei (Fig. 3A). of Viçosa (Minas Gerais state) and Boituva (São Paulo Although the structural organization of the ileal wall state) and transported to the laboratory where they were was similar in all of the species examined here, there killed by freezing. The bees were subsequently dissected were some variations in epithelial cell shape, position using a stereomicroscope and the digestive tract was of the nucleus, degree of chromatin condensation and removed, immersed in insect saline solution (0.1 M NaCl, cuticular thickness. Thus, for example, Apis mellifera, 0.1 M Na2HPO4, 0.1 M KH2PO4) and then transferred to Zamboni solution [40] for 4 h. After fixation, the hindgut Cephalotrigona capitata, Exomalopsis auropilosa, was isolated and the length of the ileum was measured Melipona rufiventris, M. compressipes, M. eburnea, using a micrometer eyepiece (Olympus). Nannotrigona testaceicornis, Partamona helleri, For histological analysis, the tissues were dehydrated Paratrigona lineata, Plebeia remota, and Tetrapedia in an ethanol series, embedded in glycol metacrylate (JB4 nigritarsis had cuboidal epithelial cells (Fig. 3A,B) EMS historesin, Warrington, PA, USA), sectioned into 4- while the other species had columnar cells (Figs. 3C,D μm-thick sections and stained with hematoxylin and eosin and 4A,C). (HE). The Feulgen reaction was done to identify nuclear The cuticle that lined the epithelium throughout DNA [24]. The thickness of the cuticle lining the lumen of its entire length varied in thickness from 0.7 μm to the ileum was measured with a micrometer eyepiece. After 4.6 μm in M. scutellaris and Xylocopa frontalis, fixation and dehydration, another set of ilea was transferred respectively (Table 2). In the epithelial cells of to hexamethyldisilazane for 5 min, air dried, sputtered with Augochloropsis sp., Melipona capixaba and M. gold, and examined with a LEO VP1430 scanning electron scutellaris, the nucleus was located centrally (Fig. microscope operated at 15 kV. 3C), while in the other species it was located in the The size of the bees was determined by measuring the intertegular distance (distance between the forewing basal portion of the cells (Fig. 4A). In Augochloropsis tegulae) [5] with a micrometer eyepiece. The morphometric sp., Bombus morio, Centris violacea, Epicharis flava, data for 30 species (Table 2) were analyzed by analysis Euglossa mandibularis, Exomalopsis auropilosa, of covariance (ANCOVA) to determine the correlation Friesomelitta varia, Geotrigona sp., Megachile spp., between body size, ileal length and degree of sociality. Megalopta sp., Paratrigona lineata and Xylocopa frontalis the chromatin was decondensed (Figs. 3D RESULTS and 4A,B), although in many other species some The ileum corresponded to the proximal portion regions of condensed chromatin were seen (Figs. of the hindgut, with the transition from the distal 3A-C and 4C). The Feulgen reaction showed that the midgut to the ileum being marked by a valve located nucleus had many regions of condensed chromatin, just above the opening of the Malpighian tubules (Fig. with the cytoplasm containing numerous vacuoles 1A,B). The ileal lumen was lined by a cuticle that of different sizes, the presence of which deformed extended throughout its entire length and included the nucleus (Figs. 3A,B and 4B,C). These vacuoles the rectum (Fig. 1C,D). The posterior distal region of occurred throughout the entire length of the ileum, the ileum protruded into the rectum where it formed except in A. mellifera, E. mandibularis, F. varia, a valve-like structure (Fig. 1C). Transverse sections Melipona quadrifasciata anthidioides, M. bicolor showed that the epithelium consisted of 4-6 folds and P. helleri, in which they accumulated in the that bulged into the lumen (Fig. 2A). Externally, posterior half of the organ. Apical plasma membrane there was a single layer of circular muscle formed infoldings that reached the medium portion of the by several muscle fibers (Fig. 2C,D), and interposed cell were also observed (Fig. 3A). between this layer and the ileal epithelium was a The size of the ileum generally increased with body distinct subepithelial space that was larger at the base size (Table 2) (ANCOVA: F1.27 = 64.39; p < 0.001), of the epithelial folds where the muscle layer was but there was no relationship with the degree of sociality interrupted to allow entrance of the tracheae (Fig. (ANCOVA: F1.27 = 0.86; p < 0.361) (Fig. 5). Braz. J. morphol. Sci. (2006) 23(3-4), 405-413 Histology of the ileum in bees 407 Table 1. List of the taxa studied. Family Tribe Species Apidae Apini Apis mellifera Linnaeus, 1758 Meliponini Cephalotrigona capitata (Smith, 1854) Frieseomelitta flavicornis (Fabricius, 1798) Frieseomelitta