Lepidopteran Larval Midgut During Prepupal Instar: Digestion or Self-Digestion?

[Autophagy 3:6, 630-631; November/December 2007]; ©2007 Landes Bioscience

Addendum

Digestion or Self-Digestion? Gianluca Tettamanti1,* Annalisa Grimaldi1 Francesco Pennacchio2 M. de Eguileor1

Original manuscript submitted: 08/14/07 Manuscript accepted: 08/20/07 Previously published online as an Autophagy E-publication: http://www.landesbioscience.com/journals/autophagy/article/4908

Key words apoptosis, autophagy, Heliothis virescens, larval midgut replacement, Lepidoptera, regenerative stem cells, caspase-3 Acknowledgements

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Addendum to:

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Programmed Cell Death and Stem Cell Differentiation are Responsible for Midgut Replacement in Heliothis virescens During Prepupal Instar

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G. Tettamanti, A. Grimaldi, M. Casartelli, E. Ambrosetti, B. Ponti, T. Congiu, R. Ferrarese, M.L. Rivas-Pena, F. Pennacchio and M.D. Eguileor

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Cell Tissue Res 2007; In press

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Programmed cell death (PCD) events are of key‑importance in body remodelling during insect development, to eliminate those tissues and organs that are functional only in embryonic or larval stages. Although apoptotic PCD has been recognized to be a widespread phenomenon in several insect species, more recently autophagic PCD has also been reported as an important mechanism for suppression of large cell populations,1,2 and its impairment has been proven to cause dramatic effects on development and survival of the insect.3 Lepidoptera are the most widely used species for the study of autophagy during metamorphosis, as their amenable size and the available molecular background information on physiological/endocrine regulation of development make them ideal models to elucidate the molecular pathways underlying PCD. Among the most precocious changes during transition to the adult stage, the replacement of the larval gut is undoubtedly one of the most evident modifications that can be observed. However, so far, this process has been only incompletely characterized. In order to fill this gap, we have recently studied the midgut replacement process during the larval‑pupal transition in mature (5th instar) larvae of the tobacco budworm, Heliothis virescens, trying to shed some light on the occurring cell death mechanisms.4 Our findings demonstrate that the proliferation of regenerative stem cells is responsible for the formation of a new midgut epithelium, while the destruction of the larval midgut epithelium is accomplished by the combined action of apoptosis, as already reported by Parthasarathy and Palli,5 and autophagy. Apoptosis starts quite precociously and persists for the whole duration of the gut replacement process, but is observed only in a limited number of cells. In contrast, autophagy represents the most prominent PCD process, responsible for the loss of the large majority of the midgut cells, which starts just after the cessation of feeding and progressively increases, until the larval epithelium is completely digested. The occurrence of this self‑digestion process is supported not only by the presence of the typical morphological characters, but also by the detection of a striking increase of lysosomal enzymes.6,7 At early stages of the midgut replacement process, this PCD form could be, in theory, activated as a starvation‑induced protective mechanism, enabling the prepupa to cope with food deprivation and to maintain cell homeostasis, by providing an alternative energy source.8,9 However, the autophagic burst in H. virescens midgut takes place only after the 20‑hydroxyecdysone commitment peak,10 suggesting that this PCD modality is finely

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This work was supported by FAR 20062007 (University of Insubria) to G.T. and by MIUR-FIRB-COFIN Grants no. RBNE01YXA8/2004077251.

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*Correspondence to: Gianluca Tettamanti; Department of Structural and Functional Biology; University of Insubria; Via J.H. Dunant 3; Varese 21100 Italy; Tel.: 0039.0332.421312; Fax: 0039.0332.421300; Email: gianluca.tettamanti@ uninsubria.it

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2Department of Agricultural Entomology and Zoology “F. Silvestri”; University of Napoli “Federico II”; Portici, Italy

Programmed cell death (PCD) is crucial in body restructuring during metamorphosis of holometabolous insects (those that have a pupal stage between the final larval and adult stages). Besides apoptosis, an increasing body of evidence indicates that in several insect species programmed autophagy also plays a key role in these developmental processes. We have recently characterized the midgut replacement process in Heliothis virescens larva, during the prepupal phase, responsible for the formation of a new pupal midgut. We found that the elimination of the old larval midgut epithelium is obtained by a combination of apoptotic and autophagic events. In particular, autophagic PCD completely digests decaying tissues, and provides nutrients that are rapidly absorbed by the newly formed epithelium, which is apparently functional at this early stage. The presence of both apoptosis and autophagy in the replacement of midgut cells in Lepidoptera offers the opportunity to investigate the functional peculiarities of these PCD modalities and if they share any molecular mechanism, which may account for possible cross‑talk between them.

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of Structural and Functional Biology; University of Insubria;

Varese, Italy

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©2007 LANDES BIOSCIENCE.BiDO NOT DISTRIBUTE.

Lepidopteran Larval Midgut During Prepupal Instar

Autophagy

2007; Vol. 3 Issue 6

Autophagy in H. virescens Larvae

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11. Rusten TE, Lindmo K, Juhasz G, Sass M, Seglen PO, Brech A, Stenmark H. Programmed autophagy in the Drosophila fat body is induced by ecdysone through regulation of the PI3K pathway. Dev Cell 2004; 7:179‑92. 12. Uwo MF, Ui‑Tei K, Park P, Takeda M. Replacement of midgut epithelium in the greater wax moth, Galleria mellonela, during larval‑pupal moult. Cell Tissue Res 2002; 308:319‑31. 13. Thummel CS. Steroid‑triggered death by autophagy. Bioessays 2001; 23:677‑82. 14. Tettamanti G, Malagoli D, Marchesini E, Congiu T, de Eguileor M, Ottaviani E. Oligomycin A induces autophagy in the IPLB‑LdFB insect cell line. Cell Tissue Res 2006; 326:179‑86. 15. Hoffman KL, Weeks JC. Role of caspases and mitochondria in the steroid‑induced programmed cell death of a motoneuron during metamorphosis. Dev Biol 2001; 229:517‑36. 16. Akdemir F, Farkas R, Chen P, Juhasz G, Medved’ova L, Sass M, Wang L, Wang X, Chittaranjan S, Gorski SM, Rodriguez A, Abrams JM. Autophagy occurs upstream or parallel to the apoptosome during histolytic cell death. Development 2006; 133:1457‑65. 17. Martin DN, Baehrecke EH. Caspases function in autophagic programmed cell death in Drosophila. Development 2004; 131:275‑84. 18. Loeb MJ, Martin PA, Narang N, Hakim RS, Goto S, Takeda M. Control of life, death, and differentiation in cultured midgut cells of the lepidopteran, Heliothis virescens. In Vitro Cell Dev Biol Anim 2001; 37:348‑52. 19. Loeb M, Hakim RS. Insect midgut epithelium in vitro: An insect stem cell system. J Insect Physiol 1996; 42:1103‑11. 20. Loeb MJ, Clark EA, Blackburn M, Hakim RS, Elsen K, Smagghe G. Stem cells from midguts of Lepidopteran larvae: Clues to the regulation of stem cell fate. Arch Insect Biochem Physiol 2003; 53:186‑98.

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tuned by developmentally‑related physiological changes, as observed also in Drosophila.11 Whatever is the trigger for autophagic PCD in the H. virescens midgut, it seems clear that the nutrients released by the suppressed midgut cells are recycled, thus providing a considerable metabolic support to the development of the organism, in the absence of food intake during the pupal period. This recycling action of nutrients is supported by the morpho‑functional features of the newly formed midgut epithelium, which shows digestive and absorptive functions, as it is endowed with membrane transporters and hydrolytic enzymes. It is worth noting that the concurrent presence of apoptosis and autophagy during the midgut replacement process in H. virescens could be more complex than a simple co-occurrence of two distinct PCD processes although this scenario might be hardly ascertainable, since apoptosis in insects shows peculiar morphological features, which hinder an unequivocal categorization of dying cells.12 For this reason, further investigations are needed to correctly define the real occurrence of apoptosis in cells showing condensed heterochromatic nuclei at an advanced stage of degeneration.13,14 Moreover, the cleaved caspase‑3 staining pattern detected at mid stages of the midgut replacement process could be either due to cells prompted from the beginning to undergo apoptosis, that will become TUNEL positive at later stages, or to cells in which autophagy is converted into a pro‑death mechanism,15‑17 which would indicate a possible cross‑talk between the two pathways. Thus, a more detailed time course analysis of the two observed PCD events is required, trying also to elucidate any possible functional interaction between the pathways controlling them. This combined intervention of two different PCD processes in the midgut epithelium of Lepidoptera at the larval‑pupal transition provides a powerful experimental model system to identify apoptosis‑ and autophagy‑specific genes and to dissect the regulatory pathways shared by these two PCD forms. Moreover, the proposed study approach in vivo could be profitably complemented by in vitro observations, which are made possible by well established culture systems of midgut cells18‑20 and other available lepidopteran cell lines, in which apoptosis and autophagy can be simultaneously induced by exogenous stimuli.14,18

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