Abstract
The honeybee Apis mellifera has ecological and economic importance; however, it experiences a population decline, perhaps due to exposure to toxic compounds, which are excreted by Malpighian tubules. During metamorphosis of A. mellifera, the Malpighian tubules degenerate and are formed de novo. The objective of this work was to verify the cellular events of the Malpighian tubule renewal in the metamorphosis, which are the gradual steps of cell remodeling, determining different cell types and their roles in the excretory activity in A. mellifera. Immunofluorescence and ultrastructural analyses showed that the cells of the larval Malpighian tubules degenerate by apoptosis and autophagy, and the new Malpighian tubules are formed by cell proliferation. The ultrastructure of the cells in the Malpighian tubules suggest that cellular remodeling only occurs from dark-brown-eyed pupae, indicating the onset of excretion activity in pupal Malpighian tubules. In adult forager workers, two cell types occur in the Malpighian tubules, one with ultrastructural features (abundance of mitochondria, vacuoles, microvilli, and narrow basal labyrinth) for primary urine production and another cell type with dilated basal labyrinth, long microvilli, and absence of spherocrystals, which suggest a role in primary urine re-absorpotion. This study suggests that during the metamorphosis, Malpighian tubules are non-functional until the light-brown-eyed pupae, indicating that A. mellifera may be more vulnerable to toxic compounds at early pupal stages. In addition, cell ultrastructure suggests that the Malpighian tubules may be functional from dark-brown-eyed pupae and acquire greater complexity in the forager worker bee.
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Acknowledgements
We want to thank the Nucleus of Microscopy and Microanalysis (NMM) and technicians of the Central Apiary at the Universidade Federal de Viçosa for technical assistance.
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This study was funded by Brazilian research agencies Conselho Nacional de Desenvolvimento Científico e Tecnológico CNPq (grant number 3015165/2013-5), Coordenação de Aperfeiçoamento de Pessoal de Nível Supeiror CAPES and Fundação de Amparo à Pesquisa do Estado de Minas Gerais FAPEMIG (grant number APQ-00508-16).
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Supplementary 1
Micrographs of the Malpighian tubules of Apis mellifera incubated only with secondary antibody anti-rabbit IgG-FITC by immunofluorescence (negative control). a L5S larvae with Malpighian tubules cells negative (absence of green fluorescence) and b white-eyed pupae with Malpighian tubules cells negative (absence of green fluorescence). Note cell nucleus (red). (GIF 39 kb)
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Suplementary 2
Micrographs of the Malpighian tubules of Apis mellifera blocked with normal non immune serum and incubated only with secondary antibody anti-rabbit IgG-FITC by immunofluorescence (negative control). L5S larvae with Malpighian tubule cells negative (absence of green fluorescence). Note the larger nuclei (red) in the cells of the Malpighian tubules in regeneration (arrows) and smaller nuclei (red) in the cells of the newly formed Malpighian tubules (asterisks). (GIF 22 kb)
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Gonçalves, W.G., Fernandes, K.M., Santana, W.C. et al. Post-embryonic development of the Malpighian tubules in Apis mellifera (Hymenoptera) workers: morphology, remodeling, apoptosis, and cell proliferation. Protoplasma 255, 585–599 (2018). https://doi.org/10.1007/s00709-017-1171-3
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DOI: https://doi.org/10.1007/s00709-017-1171-3