Brain neurosecretory cell and ecdysiotropin activity of the non-diapausing,pre-diapausing and diapausing southwestern corn borer, Diatraea grandiosella Dyar

The location and number of brain neurosecretory cells were studied in the larval southwestern corn borer. One posterior, two median and two lateral groups of paraldehyde-fuchsin positive cells were found in each cerebral hemisphere.Implantation of brain parts containing different groups of neurosecretory cells revealed that the median neurosecretory cells contained higher ecdysiotropic activity than the other cell groups. In vitro culture of ecdysial gland with brain or brain-parts extract showed also that the median neurosecretory cells contained much higher ecdysiotropic activity than other neurosecretory cells. To estimate the ecdysiotropic activity of pre-diapausing 6th instar larvae, their brain or brain extract was incubated in culture medium containing an ecdysial gland from a day-4 last-instar non-diapausing larva. Data showed that the ecdysiotropic activity in the pre-diapausing larvae was far lower than in non-diapausing and diapausing larvae.     

Cell death and tissue reorganization in Rhynchosciara americana (Sciaridae: Diptera) metamorphosis and their relation to molting hormone titers

Programmed cell death (PCD) is a focal topic for understanding processes underlying metamorphosis in insects, especially so in holometabolous orders. During adult morphogenesis it allows for the elimination of larva-specific tissues and the reorganization of others for their functionalities in adult life. In Rhynchosciara, this PCD process could be classified as autophagic cell death, yet the expression of apoptosis-related genes and certain morphological aspects suggest that processes, autophagy and apoptosis may be involved. Aiming to reveal the morphological changes that salivary gland and fat body cells undergo during metamorphosis we conducted microscopy analyses to detect chromatin condensation and fragmentation, as well as alterations in the cytoplasm of late pupal tissues of Rhynchosciara americana. Transmission electron microscopy and confocal microscopy revealed cells in variable stages of death. By analyzing the morphological structure of the salivary gland we observed the presence of cells with autophagic vacuoles and apoptotic bodies and DNA fragmentation was confirmed with the TUNEL assay in salivary gland. The reorganization of fat body occurs with discrete detection of cell death by TUNEL assay. However, both salivary gland histolysis and fat body reorganization occur under control of the hormone ecdysone.     

Identification,Source, and Metabolism of N-Ethylglutamate in Escherichia coli

N-Ethylglutamate (NEG) was detected in Escherichia coli BL21 cells grown on LB broth, and it was found to occur at a concentration of ∼4 mM in these cells under these conditions. The same cells grown on M9 glucose medium contained no detectable amount of NEG. Analysis of the LB broth showed the presence of NEG, a compound never before reported as a natural product. Isotope dilution analysis showed that it occurred at a concentration of 160 μM in LB broth. Analyses of yeast extract and tryptone, the organic components of LB broth, both showed the presence NEG. It was demonstrated that NEG can be generated during the autolysis of the yeast used in the preparation of the yeast extract. Growth of these E. coli cells in LB broth prepared in deuterated water showed no incorporation of deuterium into NEG, demonstrating that E. coli cells did not generate the NEG. Cell growth rates were not affected by the addition of 5 mM NEG to either LB or M9 glucose medium. l-[ethyl-²H₄]NEG was found to be readily incorporated into the cells and metabolized by the cells. From these results, it was concluded that all of the NEG present in the cells was taken up from the medium. NEG could serve as the sole nitrogen source for E. coli when grown on M9 glucose medium in the presence of glucose but could not serve as the sole carbon source on M9 medium in the absence of glucose.During work on developing methods for the analysis of the amino acids generated by recombinant archaeal mutases, I developed procedures for the recovery and analysis of the free amino acids present in cell extracts of Escherichia coli. When these methods were applied to analysis of E. coli grown on LB broth, I always found a large amount of an unknown amino acid. Here I report on the identification of this amino acid as N-ethylglutamate (NEG). NEG has never been reported as a natural product. I demonstrate that NEG is readily taken up by E. coli and can serve as the sole source of nitrogen when the cells are grown on M9 glucose medium.     

The hormonal pathway controlling cell death during metamorphosis in a hemimetabolous insect

Metamorphosis in holometabolous insects is mainly based on the destruction of larval tissues. Intensive research in Drosophila melanogaster, a model of holometabolan metamorphosis, has shown that the steroid hormone 20-hydroxyecdysone (20E) signals cell death of larval tissues during metamorphosis. However, D. melanogaster shows a highly derived type of development and the mechanisms regulating apoptosis may not be representative in the insect class context. Unfortunately, no functional studies have been carried out to address whether the mechanisms controlling cell death are present in more basal hemimetabolous species. To address this, we have analyzed the apoptosis of the prothoracic gland of the cockroach Blattella germanica, which undergoes stage-specific degeneration just after the imaginal molt. Here, we first show that B. germanica has two inhibitor of apoptosis (IAP) proteins and that one of them, BgIAP1, is continuously required to ensure tissue viability, including that of the prothoracic gland, during nymphal development. Moreover, we demonstrate that the degeneration of the prothoracic gland is controlled by a complex 20E-triggered hierarchy of nuclear receptors converging in the strong activation of the death-inducer Fushi tarazu-factor 1 (BgFTZ-F1) during the nymphal-adult transition. Finally, we have also shown that prothoracic gland degeneration is effectively prevented by the presence of juvenile hormone (JH). Given the relevance of cell death in the metamorphic process, the characterization of the molecular mechanisms regulating apoptosis in hemimetabolous insects would allow to help elucidate how metamorphosis has evolved from less to more derived insect species.     

Interactions of the ring gland, overies, and juvenile hormone with brain neurosecretory cells in Musca domestica.

The staining intensity (median neurosecretory cell index) of the median neurosecretory cells (MNC) in Musca domestica increased as oögenesis progressed from stages 2 to 10. The amount of neurosecretory material within the MNC was dependent upon the presence of ovaries with developing or mature follicles. Ovariectomized flies had a median neurosecretory index that was 50 per cent less than that of control flies with mature eggs. In addition, we found that ring gland removal decreased the staining frequency of three different neurosecretory cell groups; increased staining frequency in another; increased the amount of neurosecretory material within the MNC fibre tract; increased the cytoplasmic area of types A and A′ MNC. Furthermore, neither the juvenile hormone analogue nor the ring gland had a direct effect on the median neurosecretory cell index but did influence neurosecretory activity indirectly by activating the ovaries. We hypothesize that an ovarian hormone—the oöstatic hormone—regulates either the release from or synthesis of neurosecretory material within the MNC.     

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 appearences are common for both cell death types. Autophagy features are also exhibited during the prepupal period. One of the lysosomal marker enzymes, acid phosphatase, levels 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 only apoptotic cell death not enough for whole gland degeneration and that more effective degeneration occurs with this cooperation.

Addendum to: Goncu E, Parlak O. Morphological changes and patterns of ecdysone receptor B1 immunolocalization in the anterior silk gland undergoing programmed cell death in the silkworm, Bombyx mori. Acta Histochem 2008; In press.     

Ecdysone signaling at metamorphosis triggers apoptosis of Drosophila abdominal muscles

One of the most dramatic examples of programmed cell death occurs during Drosophila metamorphosis, when most of the larval tissues are destroyed in a process termed histolysis. Much of our understanding of this process comes from analyses of salivary gland and midgut cell death. In contrast, relatively little is known about the degradation of the larval musculature. Here, we analyze the programmed destruction of the abdominal dorsal exterior oblique muscle (DEOM) which occurs during the first 24 h of metamorphosis. We find that ecdysone signaling through Ecdysone receptor isoform B1 is required cell autonomously for the muscle death. Furthermore, we show that the orphan nuclear receptor FTZ-F1, opposed by another nuclear receptor, HR39, plays a critical role in the timing of DEOM histolysis. Finally, we show that unlike the histolysis of salivary gland and midgut, abdominal muscle death occurs by apoptosis, and does not require autophagy. Thus, there is no set rule as to the role of autophagy and apoptosis during Drosophila histolysis.     

Protein synthesis,DNA degradation,and morphological changes during programmed cell death in labial glands of Manduca sexta

Labial glands of the tobacco hornworm Manduca sexta (Lepidoptera: Sphingiidae), homologues of Drosophila salivary glands, undergo programmed cell death (PCD) in a 4-day period during larva-to-pupa metamorphosis. The programmed death of the labial gland was examined by electron microscopy and measurement of protein synthesis as well as measurement of DNA synthesis, end-labeling of single strand breaks, and pulsed-field gel electrophoresis. One of the earliest changes observed is a sharp drop in synthesis of most proteins, coupled with synthesis of a glycine-rich protein, reminiscent of silk-like proteins. From a morphological standpoint, during the earliest phases the most prominent changes are the formation of small autophagic vacuoles containing ribosomes and an apparent focal dissolution of the membranes of the endoplasmic reticulum, whereas later changes include differing destruction at the lumenal and basal surfaces of the cell and erosion of the basement membrane. By the fourth day of metamorphosis, individual cells become rapidly vacuolated in a cell-independent manner. In the vacuolated cells on day 3, chromatin begins to coalesce. It is at this period that unequivocal nucleosomal ladders are seen and end-labeling in situ or electrophoretic techniques document single or double-strand breaks, respectively. DNA synthesis ceases shortly after the molt to the fifth instar, as detected by incorporation of tritiated thymidine and weak TUNEL labeling. Large size fragments of DNA are seen shortly after DNA synthesis ceases and thence throughout the instar, raising the possibility of potential limitations built into the cells before their final collapse. Dev. Genet. 21:249–257, 1997. © 1997 Wiley-Liss, Inc.     

Comparison of structurally analogous allatotoxins on the molting and morphogenesis of Rhodnius prolixus,and the reversal of ecdysial stasis by ecdysone

The allatotoxic effect of 3-ethoxy-4-methoxy-6-iso-pentenylphenol on nymphal molting and metamorphosis of Rhodnius prolixus was examined. Continuous contact treatment with IPP induced the formation of precocious adults and retarded molting or initiated a permanent ecdysial stasis. Insects treated with 7-ethoxy-6-methoxy-2,2-dimethylchrornene were similarly affected. Ecdysone given orally counteracted the ecdysial stasis and also reduced the duration of the molting delay caused by IPP.     

Cellular metamorphosis. II. The larval labial gland duct and its prospective adult fates in the tobacco hornworm.

The larval labial gland of the sphingid moth, Manduca sexta, produces a viscous secretion, presumably a lubricant, facilitating the burrowing which precedes pupation. During metamorphosis, the gland transforms into a salivary organ, producing an invertase-rich digestive secretion. The single-cell type found in the duct of the larval gland transforms into the four structurally and functionally distinct cell types found in the four sequentially arranged secretory and conductive regions of the adult salivary gland. Surgical experiments were performed to study the prospective fates of different parts of the larval gland. The glands were bisected and one or both fragments were left in situ to undergo metamorphosis. In addition, fragments of the larval gland were implanted in pupal hosts and went through metamorphosis free of their prior attachments. The four linearly arrayed adult regions originate from correspondingly positioned areas in the larval duct.     

Implantation Serine Proteinases heterodimerize and are critical in hatching and implantation

Background

Metamorphosis is a complex, highly conserved and strictly regulated development process that involves the programmed cell death of obsolete larval organs. Here we show a novel functional role for the aspartic proteinase cathepsin D during insect metamorphosis.

Results

Cathepsin D of the silkworm Bombyx mori (BmCatD) was ecdysone-induced, differentially and spatially expressed in the larval fat body of the final instar and in the larval gut of pupal stage, and its expression led to programmed cell death. Furthermore, BmCatD was highly induced in the fat body of baculovirus-infected B. mori larvae, suggesting that this gene is involved in the induction of metamorphosis of host insects infected with baculovirus. RNA interference (RNAi)-mediated BmCatD knock-down inhibited programmed cell death of the larval fat body, resulting in the arrest of larval-pupal transformation. BmCatD RNAi also inhibited the programmed cell death of larval gut during pupal stage.

Conclusion

Based on these results, we concluded that BmCatD is critically involved in the programmed cell death of the larval fat body and larval gut in silkworm metamorphosis.     

Brain hormone carrier haemocytes in the moth Monema flavescens

The movement of neurosecretory substances released from the neurosecretory B cell in the pars intercerebralis to the haemolymph was examined with the progress of the termination of diapause in the slug moth pharate pupa, Monema flavescens.The injection of precipitates in the haemolymph of the pharate pupa just before the termination of diapause into diapausing pharate pupae reduced the numbers of days required for them to pupate. In the precipitates, seven types of haemocytes were present. The number of haemocytes, especially the granular cell, increased just before the termination of diapause. AF and CHP positive substances not detected in the haemocytes of diapausing pharate pupae appeared in the granular cells just before the termination of diapause. The period also coincided well with the releasing period of the neurosecretory B cell. Histological examination showed that granular haemocytes gathered around the pars intercerebralis at this period and exchange of neurosecretory substances occurred between granular haemocytes and neurosecretory B cells. Then granular haemocytes migrated to the region of the prothoracic gland. From digestion tests of the neurosecretory substances with rabbit serum and from the implantation tests of the neuroendocrine system, the substances detected in both the neurosecretory B cell and the granular haemocytes seemed to be the same. The dye injection caused a delay in larval-pupal ecdysis emergence. Droplets of black ink are incorporated into the granular haemocytes. This seems to be caused by blocking of the transport of neurosecretory substances released from cytoplasmic processes of the neurosecretory B cell.From these experiments, it is suggested that neurosecretory substances of the prothoracotropic hormone are transported to the prothoracic gland, along with granular haemocytes, after being released directly from the neurosecretory B cell to the haemolymph.     

Penetration of the cuticular layers of elaterid larvae (Coleoptera) by the fungus Metarrhizium anisopliae, and notes on a bacterial invasion

The penetrant hyphae of Metarrhizium anisopliae in the exuvial cuticle of a molting wireworm can form secondary appressoria on the developing new cuticle. From these a new penetrant fungal apparatus can develop through the new cuticle toward the body cavity. The penetrant fungal apparatus in the ecdysial space of the host does not appear to be affected by the histolytic enzymes in the wireworm molting fluid. A mucoidlike substance that envelopes the fungus in the ecdysial space may be, in part, the protective mechanism involved. Bacteria from the soil often invade the ecdysial space of molting wireworms that have difficulty in shedding their exuvia. Pseudomonas aeruginosa can histolyze the proteinaceous exocuticle of the exuvium, the ecdysial membrane, and the dense inner epicuticle of the new cuticle, but not the epicuticle of the exuvium, when it invades the ecdysial space of a molting wireworm.     

Identification of factors that function in Drosophila salivary gland cell death during development using proteomics

Proteasome inhibitors induce cell death and are used in cancer therapy, but little is known about the relationship between proteasome impairment and cell death under normal physiological conditions. Here, we investigate the relationship between proteasome function and larval salivary gland cell death during development in Drosophila. Drosophila larval salivary gland cells undergo synchronized programmed cell death requiring both caspases and autophagy (Atg) genes during development. Here, we show that ubiquitin proteasome system (UPS) function is reduced during normal salivary gland cell death, and that ectopic proteasome impairment in salivary gland cells leads to early DNA fragmentation and salivary gland condensation in vivo. Shotgun proteomic analyses of purified dying salivary glands identified the UPS as the top category of proteins enriched, suggesting a possible compensatory induction of these factors to maintain proteolysis during cell death. We compared the proteome following ectopic proteasome impairment to the proteome during developmental cell death in salivary gland cells. Proteins that were enriched in both populations of cells were screened for their function in salivary gland degradation using RNAi knockdown. We identified several factors, including trol, a novel gene CG11880, and the cop9 signalsome component cop9 signalsome 6, as required for Drosophila larval salivary gland degradation.     

The formation of the ecdysial droplets and the ecdysial membrane in an insect

Insect cuticle forms as a result of overlapping sequences of two kinds of process, those involving vesicles of the Golgi complex, and those related to transport through and/or assembly at the apical plasma membrane. The ecdysial droplets are the last layer of old cuticle to be deposited before ecdysis and form from the contents of secretory vesicles from Golgi complexes. Ecdysial droplets and secretory vesicles both stain with PTA and react with silver hexamine after oxidation with periodic acid. The vesicles discharge in localized apical areas devoid of microvilli where they accumulate as droplets measuring about 3 [ x 1 [. The. droplets span the last few lamellae of the endocuticle which becomes the ecdysial membrane. They dissolve to leave the ecdysial membrane full of holes at the time that the rest of the old cuticle is digested.     

Influence of thyroid hormones on neuronal death and differentiation in larval Rana pipiens.

The sphingid moth, Manduca sexta, typically passes through five larval instars, a pupal, and an adult stage. The larval labial glands secrete silk in the first instar and a viscous lubricant in the fifth. During metamorphosis the glands develop into salivary organs which produce an invertase-rich secretion. In normal development, the uniform population of cells in the duct of the larval gland transforms into the four sequentially arranged regions of secretory and conductive cells of the adult gland. In order to determine when competence to form the adult gland is established, fragments of labial gland ducts from first through fifth instar larvae were implanted into pupae. These gland fragments underwent metamorphosis with their hosts, passing through the same developmental phases. Glands from as early as the first instar were competent to form histologically and functionally normal adult regions. In later instars, transplants of measured fragments demonstrated that larval cells were programmed in situ to develop into the four adult cell types.     

家蚕蛹变态期丝腺组织的退化与细胞凋亡特征

利用形态学观察方法、分子生物学检测方法以及20-羟基蜕皮酮(20-hydroxyecdysone)和放线菌酮(cycloheximide)体外培养方法, 研究了家蚕Bombyx mori 蛹变态期丝腺组织的退化与细胞凋亡特征。显微镜的观察显示家蚕丝腺的逐渐退化发生在吐丝期间。DNA梯度电泳的分析表明程序性细胞死亡(programmed cell death)可能伴随发生在丝腺的退化过程中。在离体培养条件下, 用20-羟基蜕皮酮处理5龄第6天幼虫的丝腺, 导致的细胞凋亡提前于对照, 提示在进入蛹变态期前, 20-羟基蜕皮酮提早激发了介导家蚕丝腺细胞凋亡与水解机制的遗传调控级联系统。上述结果表明, 20-羟基蜕皮酮能够诱导家蚕丝腺组织在蛹变态期发生程序性细胞死亡。