Elsevier

Micron

Volume 37, Issue 2, February 2006, Pages 161-168
Micron

The regenerative cells during the metamorphosis in the midgut of bees

https://doi.org/10.1016/j.micron.2005.07.003Get rights and content

Abstract

The midgut epithelium of bees is formed by the digestive cells, responsible for enzyme secretion and nutrient absorption and for small regenerative cells that are placed in nests scattered among the digestive cells. During metamorphosis, the larval midgut epithelium degenerates and a new adult midgut epithelium is built during larval differentiation of regenerative cells. The present work focuses on the midgut epithelial modifications during the post-embryonic development of the stingless bee Melipona quadrifasciata anthidioides worker and the occurrence of regenerative cell proliferation during midgut metamorphosis in order to test the hypothesis that adult midgut epithelium of worker bees results from regenerative cell proliferation during the pupal stage. Regenerative cell proliferation was detected during larval lifespan. Larval aging is followed by an increase in the number and the size of the nests of regenerative cells. Larval epithelium degeneration begins 2 days after the start of defecation process and in this period the nests of regenerative cells are in contact by means of cytoplasmic extension which have many septate desmosomes and gap junctions. The BrdU immunoreactive regenerative cells were found in the prepupae 12 h after BrdU injection, suggesting that regenerative cell population increase during this larval period. Regenerative cell proliferation results in the increase of the regenerative cell population and not in the formation of new digestive cells because the proliferation of regenerative cells would not be enough to reestablish the nests of regenerative cells and at the same time form new adult digestive cells. In this sense the hypothesis that digestive adult cells originate from regenerative cell proliferation during midgut metamorphosis in M. quadrifasciata anthidioides was rejected.

Introduction

The midgut of bees has three cell types: digestive cells that synthesize digestive enzymes and absorb the nutrients, the endocrine cells producing hormone and the regenerative cells responsible for the replacement of the former cell types.

Digestive cells in newly hatched larvae form a cylindrical or cubical epithelium acquiring granules, vacuoles and apical protrusions with aging. In the prepupae the midgut becomes narrowed and elongated, while in white eyed pupae the midgut acquires a bottle shape and the diameter increases. In pink eyed pupae the midgut diameter becomes uniform and the digestive cell apex is cast out to the lumen, but in black eyed pupae the midgut has epithelium similar to the adult (Oertel, 1930, Snodgrass, 1956, Cruz-Landim and Mello, 1970, Neves et al., 2002).

The regenerative cells have little cytoplasm and the nuclei have condensed chromatin. In the cytoplasm there are few ribosome, mitochondria, endoplasmic reticulum and Golgi apparatus profiles suggesting low metabolic activity in these cells (Hecker et al., 1971, Cruz-Landim, 1985, Cruz-Landim et al., 1996, Cruz-Landim, 1999). At metamorphosis the larval midgut epithelium is ejected and its reorganization occurs from regenerative cells. During the regenerative differentiation cells elongate in direction to the midgut lumen acquiring microvilli, followed by an increase in the nuclear and cytoplasmic volume (Werner et al., 1991, Cruz-Landim et al., 1996, Neves et al., 2003).

In Hymenoptera changes in the midgut begin in the prepupa with the larval epithelium degenerating, leaving only the basement membrane and the regenerative cells. At the same time, some cells proliferate and are scattered on the basement membrane to build the new pupal epithelium (Oertel, 1930, Snodgrass, 1956, Cruz-Landim and Mello, 1970, Gama and Cruz-Landim, 1984, Neves et al., 2002). However, during the metamorphosis of bees, regenerative cells in division had been rarely found (Oertel, 1930, Cruz-Landim and Mello, 1970, Neves et al., 2003, Cruz-Landim and Cavalcante, 2003).

Cell proliferation is easily recognized when there are cells in mitosis with condensed chromosomes. An alternative procedure for cell proliferation detection is the use of antibodies against a specific substance present in the cells that are preparing to proliferate. 5-Bromo-2′-deoxy-uridine (BrdU) is an analogous of the thymidine nucleotide incorporated to DNA during the replication process (Griffiths et al., 2000, Voet et al., 2000). In this way, antibody anti-BrdU can be used to identify cells that incorporated BrdU providing evidence for cell proliferation (Gratzner, 1982). BrdU has been used successfully in insects for detection of cell proliferation (Kirschenbaum and O'Shea, 1993, Zacharias et al., 1993, Stocker et al., 1995, Loeb and Hakim, 1996, Schimdt-Capella and Hartfelder, 1998, Vitt and Hartfelder, 1998, Cayre et al., 2000). In this work, we used this procedure to investigate the presence of cell proliferation in the midgut of the stingless bee Melipona quadrifasciata anthidioides during post-embryonic development to test the hypothesis that new digestive cells arise from regenerative cells due to mitosis.

Section snippets

Animals

Melipona quadrifasciata anthidioides were obtained from nests maintained in the Federal University of Viçosa, state of Minas Gerais, Brazil. The bees were analyzed in the following developmental stage: 27 larvae of various sizes, 24 prepupae, 54 pupae (18 white eyed pupae, 18 brown eyed pupae and 18 black eyed pupae) and eight adults (four nurse and four forager workers). The greater number of immature specimens was obtained because we injected BrdU in 30 specimens per stage and some them died

Results

In the M. quadrifasciata anthidioides larvae the midgut epithelium is formed by a single layer of digestive cells that are prismatic increasing in size with the larval development and the nests of regenerative cells are located at the base of the midgut epithelium (Fig. 1). In the young larvae the regenerative cells are spherical or oval forming isolated nests with one or two regenerative cells per nest. Some nests are found lined by a digestive cell, but mostly they are found between the basal

Discussion

The midgut reorganization of bees durin metamorphosis was studied by Oertel, 1930, Snodgrass, 1956, Cruz-Landim and Mello, 1970, Neves et al., 2002, Neves et al., 2003, Cruz-Landim and Cavalcante, 2003. However, these studies did not look at the regenerative cell nest reorganization.

The regenerative cell nests in adult of M. quadrifasciata anthidioides are equidistant, as in Apis mellifera (Raes et al., 1994). These nests are located in the deep regions of the midgut villi, created by the

Acknowledgements

We are grateful to Dr Silvia G. Pompolo and Dr Adilson A. Zacaro (Federal University of Viçosa, Brazil) for criticism and suggestions and to Dr L.A.O. Campos for criticism and for providing the bees. This work was financially supported by National Research Council (CNPq) and Minas Gerais State Research Council (FAPEMIG). G.F.M. is a graduate fellow of CNPq and C.A.N., L.A.O.C and J.E.S. are staff members of UFV and the two later are research fellow of CNPq.

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