Ji-da Dai

Metamorphosis of the corpus allatum and degeneration of the prothoracic glands during the larval-pupal-adult transformation of Drosophila melanogaster: A cytophysiological analysis of the ring gland

The degeneration of the prothoracic glands of Drosophila melanogaster during pupal-adult metamorphosis was analyzed by light microscopy, scanning, and transmission electron microscopy. The ultrastructural observations were correlated with the ability of the ring gland to synthesize ecdysteroids in vitro. The ring gland is prominent during larval life and is identifiable until just before adult eclosion but undergoes dramatic changes in location, shape, size, ultrastructure, and function during pupal-adult development. Prothoracic gland degeneration is characterized by: a gradual decrease in its ability to synthesize ecdysteroids; a decreasing quantity of smooth endoplasmic reticulum (SER) and mitochondria; the absence of intercellular channels; cytoplasmic fragmentation; and the separation of the prothoracic gland from the corpus allatum and corpus cardiacum. An ultrastructural analysis of the corpus allatum during larval-pupal-adult metamorphosis and adult life was also correlated with function, i.e., juvenile hormone biosynthesis, using a radiochemical assay of ring glands and adult corpora allata in vitro. A relatively high concentration of SER, mitochondria, and mitochondrion-scalariform junction complexes are typical features of an active corpus allatum cell. The migration of the corpus allatum from the ring gland to its position as a separate gland in the adult fly was studied in detail. The capacity of the corpus allatum to synthesize juvenile hormone is at its peak in the ring gland of the early wandering third instar larva, whereas the corpus allatum of 2-day-old female adults displayed the greatest synthetic activity during adult life. The physiological significance of the alterations in gland activity is discussed.

Programmed cell death of the prothoracic glands of Manduca sexta during pupal-adult metamorphosis

The degeneration of the prothoracic glands of Manduca sexta during pupal-adult metamorphosis was analyzed by both light and transmission electron microscopy, and correlated with a functional analysis of the ability of the prothoracic glands to synthesize ecdysteroids in vitro. Apoptosis of degenerating glandular cells was also examined by in situ indirect immunofluorescein detection of digoxigenin-labeled genomic DNA cleavage (the TUNEL method). The ability of the prothoracic gland to synthesize ecdysteroids reached a peak 4 days after pupation, decreased drastically on day 6 and then decreased further to the basal level 2 days later. Prothoracic gland degeneration was initiated on day 6 and was characterized by nuclear condensation, cytoplasmic budding, giant autophagic vacuoles, the disappearance of the smooth endoplasmic reticulum and intercellular channels, and the fragmentation of the cytoplasm into membrane-bound bodies. The cell debris of these degenerating cells was then engulfed by numerous phagocytic hemocytes. The results of the analysis of apoptosis by immunofluorescence detection are in complete accord with the histological, ultrastructural and radioimmunoassay data, suggesting that apoptosis is the basic mechanism for programmed cell death of the cells comprising these vital glands.

Immunoreactivity of neurosecretory granules in the brain-retrocerebral complex of Manduca sexta to heterologous antibodies against Bombyx prothoracicotropic hormone and bombyxin

Summary A monoclonal antibody against a synthetic pentadecapeptide corresponding to the N-terminal end of Bombyx-prothoracicotropic hormone (PTTH: 1–15) and a monoclonal antibody against a synthetic decapeptide corresponding to the N-terminal portion of the A-chain of bombyxin-1, bombyxin (1–10), were used as parallel probes to localize both molecules at the cellular and subcellular levels in the brain-retrocerebral complex of Manduca sexta. Immunohistochemical analysis revealed that molecules immunochemically similar to Bombyx-PTTH were localized in the cytoplasm of two pairs of dorso-lateral neurosecretory cells (L-NSC III) and could be traced along their axons to the corpora cardiaca (CC) and corpora allata (CA), whereas bombyxin-like material was absent from these cells and was detected only in the cytoplasm of four pairs of median neurosecretory cells (M-NSCs). Immunocytochemical labelling at the ultrastructural level using the PTTH antibody showed high specificity with the 5 nm immunogold particles ...

An ultrastructural analysis of the ecdysoneless ((l(3)ecd1ts) ring gland during the third larval instar of Drosophila melanogaster

SummaryIn the late third larval instar of Drosophila melanogaster, the prothoracic gland, an endocrine portion of the ring gland, synthesizes ecdysteroids at an accelerated rate. The resultant ecdysteroid titer peak initiates the events associated with metamorphosis. The normal prothoracic gland displays several ultrastructural features at this developmental stage that reflect increased steroidogenic activity, including extensive infoldings of the plasma membrane (membrane invaginations) and an increase in both the concentration of smooth endoplasmic reticulum (SER) (or transitional ER) and elongated mitochondria. By contrast, the prothoracic glands of larvae homozygous for a conditional larval lethal mutation, l(3)ecd1ts, not only fail to produce ecdysteroids at normal levels at the restrictive temperature (29° C), but also acquire abnormal morphological features that reflect the disruptive effects of the mutation. These abnormalities include an accumulation of lipid droplets presumed to contain sterol precursors of ecdysteroids, a disappearance of SER and a drastic reduction of membrane invaginations in the peripheral area of the cell. These morphological defects are observed in prothoracic glands dissected from larvae transferred from 18° C to 29° C approximately 24 h before observation and also within 4 h of an in vitro transfer to 29° C following dissection from wandering third instar larvae reared at 18° C. No ultrastructural abnormalities were noted in the corpus allatum portion of mutant ring glands. These observations further indicate the direct involvement of the ecd gene product in ecdysteroid synthesis and suggest a role for the gene in the proper transport of precursors to the site where they can be utilized in ecdysteroid biosynthesis.

An ultrastructural and developmental analysis of the corpus allatum of juvenile hormone deficient mutants ofDrosophila melanogaster

SummaryThe ultrastructure of the corpus allatum of theapterous mutantsap4 andap56f ofDrosophila melanogaster during larval-pupal-adult metamorphosis and adult life was correlated with the glands ability to synthesize juvenile hormone in vitro. During the early wandering period of the third instar of both mutants, a high concentration of smooth endoplasmic reticulum, mitochondria and mitochondrion-scalariform junction complexes are typical features of an active corpus allatum cell. Juvenile hormone biosynthesis by the glands is high at that time and, in fact, only slightly lower than that of wild type glands. In contrast to the wild type gland, the cells of the pupal and pharate adult corpus allatum of both mutants contains highly electron dense mitochondria with tubular cristae but no whorls of smooth endoplasmic reticulum nor glycogen clusters. The frequency and size of the lipid droplets, putatives depots of the juvenile hormone precursors, in cells of theap56f gland is a function of the insects age, but both are lower than in wild type gland cells. Juvenile hormone biosynthesis by both mutant glands remains at the basal level when compared to increased synthesis by the wild type gland. The frequency and density of lipid droplets in cells of theap4 corpus allatum are much lower than in theap56f glands. During adult life, the ultrastructural profile of theap56f corpus allatum is similar to that of the wild type gland although the in vitro production of juvenile hormone by the former is much lower than that of the wild type gland. The ultrastructural features of the adult corpus allatum ofap4 homozygotes reveal precocious degeneration and support the view that this non-vitellogenic mutant is a juvenile hormone deficient mutation.

An autoradiographic and immunocytochemical analysis of ecdysteroids and ecdysteroid binding sites in target cells of Drosophila melanogaster

Summary Ecdysteroid localization in target tissues of Drosophila melanogaster was demonstrated by the utilization of low temperature autoradiography and immunohistochemistry. Autoradiographic studies utilized high specific activity [3H]ponasterone A, an ecdysteroid with exceptional molting hormone activity in Drosophila. The data revealed a selective nuclear concentration of the labelled ligand in cells of the salivary gland, corpus allatum (source of juvenile hormone), prothoracic gland (source of ecdysone) and the central nervous system. Nuclear localization was also observed in epithelial cells of the esophagus and muscle cells of the pharynx and mandibles, etc. The specific labelling of cell nuclei was prevented by incubation of the tissue with [3H]ponasterone A in the presence of excess unlabelled ponasterone A. Nuclear localization of ponasterone A in the salivary gland was also observed when using an antiserum against ecdysteroids and an avidin-biotin-complex (ABC) detection system. The staining sh...