Role of autophagy in midgut stem cells of silkworm Bombyx mori, during larval-pupal metamorphosis.

Autophagy is a critical mechanism for the self-renewal, proliferation, and differentiation of stem cells. Bombyx mori midgut has stem cells that play a role in the regeneration of the larval epithelium in larval stages and the formation of the pupal midgut epithelium during larval-pupal metamorphosis. In this study, the role of the autophagy mechanism in midgut stem cells during the formation of the pupal midgut was investigated. For this purpose, two different doses of autophagy inhibitor chloroquine were administered to B. mori larvae on days 7 and 8 of the fifth larval stage. Morphological changes during the formation process of the pupal epithelium, expression levels of autophagy-related genes Atg8 and Atg12 in stem cells, and the amounts of lysosomal enzyme acid phosphatase were determined after the application. The obtained findings were evaluated in comparison with the control groups. Abnormalities in the formation of the pupal midgut after inhibition of autophagy showed the significance of the autophagy mechanism during this period.

Apoptotic and autophagic characteristics of perivisceral fat body remodeling of the greater wax moth Galleria mellonella and effects of juvenile hormone analog, fenoxycarb, on these processes.

In holometabolous insects, many tissues and organs such as the fat body and midgut undergo a remodeling process during metamorphosis. Larval fat body cells are eliminated by programmed cell death (PCD), while tissue cells that adapt to adult life are formed by stem cells. In this study, we analyzed the features of the remodeling period of Galleria mellonella fat body in terms of PCD types, apoptotic and autophagic cell death characteristics. Besides, the effects of juvenile hormone (JH) on these processes were evaluated under the modified hormonal conditions via applications of JH analog, fenoxycarb. Several hallmarks of apoptotic and autophagic cell death were analyzed by morphological, biochemical, and molecular methods. The results of the present study have ascertained that the degeneration process of larval cells occurs via autophagic cell death accompanied by caspase-3 activity during the pupal period and it is regulated by 20-hydroxyecdysone (20HE) mediated by ecdysone receptor B1 (EcR-B1). Increased activity of the acid phosphatase and upregulation of ATG6 and ATG8 in parallel with the formation of autophagosomes in the fat body of Galleria during the pupal period strongly indicated that autophagy was the key player in the remodeling processes.

Developmental Expression of Ecdysone-Related Genes Associated With Metamorphic Changes During Midgut Remodeling of Silkworm Bombyx mori (Lepidoptera:Bombycidae).

Steroid hormone 20-hydroxyecdysone is known as the systemic regulators of insect cells; however, how to impact the fate and function of mature and stem cells is unclear. For the first time, we report ecdysone regulatory cascades in both mature midgut cell and stem cell fractions related to developmental events by using histological, immunohistochemical, biochemical and gene expression analysis methods. Ecdysone receptor-B1 (EcR-B1) and ultraspiracle 1 (USP-1) mRNAs were detected mainly in mature cells during programmed cell death (PCD). Lowered E75A and probably BR-C Z4 in mature cells appear to provide a signal to the initiation of PCD. E74B, E75B and BR-C Z2 seem to be early response genes which are involved in preparatory phase of cell death. It is likely that ßFTZ-F1, E74A and BR-C Z1 are probably associated with execution of death. EcR-A and USP2 mRNAs were found in stem cells during remodeling processes but EcR-B1, USP1 and E74B genes imply an important role during initial phase of metamorphic events in stem cells. BHR3 mRNAs were determined abundantly in stem cells suggesting its primary role in differentiation. All of these results showed the determination the cell fate in Bombyx mori (Linnaeus) midgut depends on type of ecdysone receptor isoforms and ecdysone-related transcription factors.

The effect of the juvenile hormone analog, fenoxycarb, on ecdysone receptor B1 expression in the midgut of Bombyx mori during larval-pupal metamorphosis.

The Bombyx mori (Lepidoptera: Bombycidae) midgut undergoes remodeling during the larval-pupal metamorphosis. All metamorphic events in insects are controlled by mainly two hormones: 20-hydroxyecdysone (20E) and juvenile hormone (JH). Fenoxycarb, O-ethyl N-(2-(4-phenoxyphenoxy)-ethyl) carbamate, has been shown to be one of the most potent juvenile hormone analogs against a variety of insect species. In this study, the effect of fenoxycarb on EcR-B1 protein expression in the midgut of Bombyx mori during the remodeling processwas investigated. Fenoxycarb was topically treated to the beginning of the fifth instar Bombyx larvae. Its application prolonged the last instar and prevented metamorphic events. Analyses were performed from day 6 of the fifth instar to 24 hr after pupation in controls and to day 14 of the fifth instar in the fenoxycarb treated group. According to our results, the presence of EcR-B1 in the midguts of the fenoxycarb treated group during the feeding period suggested that EcR-B1 was involved in the functioning of larval cells and during this period fenoxycarb did not affect EcR-B1 status. Immediately after termination of the feeding stage, the amount of EcR-B1 protein increased, which indicated that it may strengthen the ecdysone signal for commitment of remodeling process. In the fenoxycarb treated group, its upregulation was delayed, which may be related to the inhibition of ecdysone secretion from the prothoracic gland.

Morphological changes and patterns of ecdysone receptor B1 immunolocalization in the anterior silk gland undergoing programmed cell death in the silkworm, Bombyx mori.

The silk gland is a specific larval tissue of Lepidopteran insects and begins to degenerate shortly before pupation. The steroid hormone ecdysone triggers the stage specific programmed cell death of the anterior silk glands during metamorphosis in the silkworm, Bombyx mori. The anterior silk gland expresses ecdysone receptors, which are involved in regulation processes in response to ecdysone. In this study, the morphological changes, immunohistochemical localization and protein levels of ecdysone receptor B1 (EcR-B1) in the anterior silk gland of B. mori were investigated during programmed cell death. Morphological changes observed during the degeneration process involve the appearance of large vacuoles, probably autophagic vacuoles, which increase in number in pupal anterior silk glands. No macrophages were found in the silk gland during the prepupal and pupal stage unlike in apoptosis, which strongly suggests that programmed cell death of the anterior silk gland is carried out by autophagy. Morphological changes of the silk glands were accompanied by changes in the immunolocalization and protein levels of EcR-B1. The differences in tissue distribution and protein levels of EcR-B1 during the programmed cell death indicate that the receptor plays a major role in the modulation and function of ecdysone activity in Bombyx anterior silk glands. Our results indicate that EcR-B1 expression may be important for the process of programmed cell death in the anterior silk glands.

Some autophagic and apoptotic features of programmed cell death in the anterior silk glands of the silkworm, Bombyx mori.

Programmed cell death has been subdivided into two major groups: apoptosis and autophagic cell death. The anterior silk gland of Bombyx mori degenerates during larval-pupal metamorphosis. Our findings indicate that two types of programmed cell death features are observed during this physiological process. During the prepupal period, pyknosis of the nucleus, cell detachment,and membrane blebbing occur and they are the first signs of programmed cell death in the anterior silk glands. According to previous studies, all of these morphological appearances are common for both cell-death types. Autophagy features are also exhibited during the prepupal period. Levels of one of the lysosomal marker enzymes-acid phosphatase-are high during this period then decrease gradually. Vacuole formation begins to appear first at the basal surface of the cell, then expands to the apical surface just before the larval pupal ecdysis. After larval-pupal ecdysis, DNA fragmentation, which is the obvious biochemical marker of apoptosis, is detected in agarose gel electrophoresis, which also shows that caspase-like enzyme activities occur during the programmed cell death process of the anterior silk glands. Apoptosis and autophagic cell death interact with each other during the degeneration process of the anterior silk gland in Bombyx mori and this interaction occurs at a late phase of cell death. We suggest that apoptotic cell death only is not enough for whole gland degeneration and that more effective degeneration occurs with this cooperation.