Different modes of programmed cell death during oogenesis of the silkmoth Bombyx mori

It is increasingly recognized that programmed cell death includes not only apoptosis and autophagy, but also other types of nonapoptotic cell death, such as paraptosis, which are all characterized by distinct morphological features. Our findings indicate that all three types of programmed cell death occur in the ovarian nurse cell cluster during late vitellogenesis (formation of the egg yolk) of Bombyx mori (Lepidoptera), whereas middle vitellogenesis is exclusively characterized by the presence of a nonapoptotic type of cell death, known as paraptosis. During middle vitellogenesis, nurse cells exhibit clearly cytoplasmic vacuolization, as revealed by ultrastructural examination performed through conventional light and transmission electron microscopy, while no signs of apoptotic or autophagic features are detectable. Moreover, nurse cells of developmental stages 7, 8 and 9 contain autophagic compartments, as well as apoptotic characteristics, such as condensed chromatin, fragmented DNA and activated caspases, as revealed by in vitro assays. We propose that paraptosis precedes both apoptosis and autophagy during vitellogenesis, since its initial activation is detectable during middle vitellogenesis, whereas no apoptotic nor autophagic features are observed. In contrast, at the late stages of Bombyx mori oogenesis, paraptosis, autophagy and apoptosis operate synergistically, resulting in a more efficient elimination of the degenerated nurse cells.

Addendum to: Mpakou VE, Nezis IP, Stravopodis DJ, Margaritis LH, Papassideri IS. Programmed cell death of the ovarian nurse cells during oogenesis of the silkmoth Bombyx mori. Dev Growth Differ 2006; 48:419–28.     

Programmed cell death of the ovarian nurse cells during oogenesis of the silkmoth Bombyx mori

In the present study, we describe the features of programmed cell death of the ovarian nurse cells occurring during vitellogenesis of the silkmoth Bombyx mori. At developmental stage 5, the nurse cells occupy one-half of the follicular volume and obtain a rather spherical shape, while the nurse cell nuclei appear large and elongated, forming impressive projections. At the following stage, stage 6, the nurse cells decrease in size and their shape becomes elliptic. The nuclei remain elongated, being also characterized by large lobes. The lobes of the ramified nurse cell nuclei seem to retain the nucleus in the center of the cell during the dumping of the nurse cell cytoplasm into the growing oocyte. At stage 7, membrane enclosed vacuoles can be easily detected into the nurse cells cytoplasm. Ultrastructural analysis and fluorescent microscopy using mono-dansyl-cadaverine staining of these vacuoles also reveal that they represent autolysosomes. Caspase activity is detected during stage 7, as it is demonstrated by using the Red-VAD-FMK staining reagent. At developmental stages 8 and 9, the nurse cells exhibit chromatin condensation, DNA fragmentation and caspase activity. Finally, during the following stage 10, the nuclear remnants are assembled into apoptotic vesicles, which, after being phagocytosed, are observed in the cytoplasm of adjacent follicle cells. We propose that apoptosis and autophagy operate synergistically during vitellogenesis of B. mori, in order to achieve an efficient and rapid clearance of the degenerated nurse cell cluster.     

Proteomic analysis of silk gland programmed cell death during metamorphosis of the silkworm Bombyx mori

The silk gland of the silkworm Bombyx mori undergoes programmed cell death (PCD) during pupal metamorphosis. On the basis of their morphological changes and the occurrence of a DNA ladder, the tissue cells were categorized into three groups: intact, committed, and dying. To identify the proteins involved in this process, we conducted a comparative proteomic analysis. Protein expression changes among the three different cell types were examined by two-dimensional gel electrophoresis. Among approximately 1000 reproducibly detected protein spots on each gel, 43 were down-regulated and 34 were up-regulated in PCD process. Mass spectrometry identified 17 differentially expressed proteins, including some well-studied proteins as well as some novel PCD related proteins, such as caspases, proteasome subunit, elongation factor, heat shock protein, and hypothetical proteins. Our results suggest that these proteins may participate in the silk gland PCD process of B. mori and, thus, provide new insights for this mechanism.     

Ecdysteroid-induced programmed cell death and cell proliferation during pupal wing development of the silkworm, Bombyx mori

The wing margin of adult wings of Lepidoptera is defined by the position of a "bordering lacuna"(BL). During adult wing development, cell proliferation and scale formation proximal to this lacuna and programmed cell death distal to the lacuna are generally observed. To determine the effect of 20-hydroxyecdysone (20E) on these events, we cultured the silkworm pupal wings with or without 20E and analyzed regional specificity for cell death by the TUNEL method and cell proliferation by 5-bromodeoxyuridine labeling. Programmed cell death was induced by 20E after 5 days of culture and was detected only in the region distal to BL. Cell proliferation after 1 day of culture and scale formation after 5 days of culture were also inducible by 20E and detected in the region proximal to BL. These results suggest that two types of pupal wing cells, which are divided by the position of the BL, respond to ecdysteroid in different manners. Higher concentrations of 20E (more than 1,000 ng/ml) repressed the scale formation, while such repression could not be observed in the peripheral cell death even with 5,000 ng/ml 20E. The ecdysteroid may work both as a trigger to make the wing margin and scales and as a developmental timer to arrange these cellular responses.     

Modes of programmed cell death during Ceratitis capitata oogenesis

In the present study, we demonstrate the existence of two distinct apoptotic patterns in nurse cells during Ceratitis capitata oogenesis. One is developmentally regulated and normally occurs during stages 12 and 13, and the other is stage specific and is sporadically observed during stages 7 and 8. The pre-apoptotic manifestation of the first pattern begins at stage 11 and is characterized by the formation of actin bundles. Subsequently, at stages 12 and 13, the nurse cell nuclei exhibit condensed chromatin and contain fragmented DNA, as revealed by TUNEL assay. The apoptotic nurse cell remnants are phagocytosed by the neighboring follicle cells at the end of oogenesis during stages 13 and 14. In the second apoptotic pattern, which occurs sporadically during stages 7 and 8, the nurse cells degenerate and are phagocytosed by the follicular epithelium that contains apoptotic cell bodies. The data presented herein, compared to previous reported results in Drosophila melanogaster and Dacus oleae (Nezis et al., 2000, 2001), strongly suggest that nurse cell apoptosis is a developmentally regulated and phylogenetically conserved mechanism in higher Dipteran. They also suggest that, the sporadic apoptotic pattern consists of a possible protective mechanism throughout oogenesis when damaged or abnormal egg chambers, are eliminated before they reach maturity.     

Heat-inducible transgenic expression in the silkmoth Bombyx mori

Germline transformation with new transposon vectors now enables causal tests of gene function via ectopic protein expression or RNA interference in non-drosophilid insects. The problem remains of how to drive the transgene expression in vivo. We employed germline transformation using the piggyBac 3xP3-EGFP vector to test whether the Drosophila heat shock hsp70 promoter will be active in the live silkworm. We modified the original vector by cloning the coding sequence for Bombyx nuclear receptor Ftz-F1 between the hsp70 promoter and the terminator. Three independent transgenic lines expressing the Pax-6-driven EGFP marker in larval and adult photoreceptors were obtained with efficiencies of up to 1.7% of fertile G0 adults that gave GFP-positive progeny. Chromosomal integration of the transposon was confirmed with inverse PCR. Heat induction of the transgenic BmFtz-F1 was proven at both the mRNA and protein levels. RT-PCR data showed that the Drosophila heat shock promoter was functional in all three transgenic lines. Although basal activity was apparent at 25 degrees C, 1 h at 42 degrees C induced BmFtz-F1 mRNA at different stages of development and in diverse tissues. The relative levels of induction differed among the transgenic lines. Northern blot hybridization detected transgenic BmFtz-F1 only after heat shock and low levels of the mRNA were still present 6 h after the heat treatment. Immunostaining of epidermis using anti-BmFtz-F1 antibody showed a clear increase of nuclear signal 90 min after a heat shock.     

Candidate pheromone binding proteins of the silkmoth Bombyx mori

Pheromone reception is thought to be mediated by pheromone binding proteins (PBPs) in the aqueous lymph of the antennal sensilla. Recent studies have shown that the only known PBP of Bombyx mori (BmorPBP1) appears to be specifically tuned to bombykol but not to bombykal, raising the question of whether additional subtypes may exist. We have identified two novel genes, which encode candidate PBPs (BmorPBP2, BmorPBP3). Comparison with PBPs from various moth species have revealed a high degree of sequence identity and the three BmorPBP-subtypes can be assigned to distinct groups within the moth PBP family. In situ hybridization revealed that BmorPBP2 and BmorPBP3 are expressed only in relatively few cells compared to the number of cells expressing BmorPBP1. Double-labeling experiments have shown that the two novel BmorPBPs are expressed in the same cells but are not co-expressed with BmorPBP1. Furthermore, unlike BmorPBP1, cells expressing the newly identified PBPs did not surround neurons containing the BmOR-1 receptor. The results indicate that BmorPBP2 and BmorPBP3 are located in sensilla types, which are different from the long sensilla trichodea.Data deposition: The sequences reported in this paper have been deposited in the EMBL database under accession nos. AM403100 (BmorPBP2) and AM403101 (BmorPBP3).     

X-ray-induced recombination during oogenesis in the silkworm (Bombyx mori L.)

Summary The physical induction of recombinational events has been studied in the female silkworm (Bombyx mori), in which crossing-over does not normally occur. Female silkworms heterozygous in the trans type of two egg-color genes,pe (V-0.0) andre (V-31.7), received a single dose of X-rays (1000 R) at various developmental stages. Then they were crossed to marked males homozygous for both genes. The results indicated that X-rays increase the occurrence of recombinational events in silkworm females from first instar larvae to late stage pupae. The spontaneous frequency of exchange type recombinants in the control series was 2.5 x 10^–5, while after irradiation the frequency of these recombinants was up to 38.8 x^10–5. The sensitive stage to X-ray-induced recombinational events was late stage larvae from fourth to fifth instar. Exchange (cross-over) type recombinants were about three times more frequent than interchange types among the 122 recombinants recovered. The biological significance of the present finding is discussed.     

Photoperiod induced developmental effects on silkmoth,Bombyx mori

Bombyx mori is most common silkmoth used for the commercial production of the silk. The different environmental conditions may affect development of silkmoth and hence the output of silkworm. Present study investigated effects of different photoperiodic conditions on the development of silkmoth. Three experiments were performed on newly hatched larvae. In experiment1, larvae were divided into three groups; group 1, 8L:16D (8 h light and 16 h dark), group 2, 12L:12D (12 h light and 12 h dark) and group 3, 16L:8D (16 h light and 8 h dark). In experiment 2, larvae were exposed to either continuous light or continuous dark. In experiment 3, larvae were divided into three groups. Each group received light of similar duration (16L:8D) and intensity (1.1 ± 0.1 PAR) but of different wavelengths; i.e. long wavelength red light (640 nm), short wavelength blue light (450 nm) and white light. Larvae were fed on young mulberry (Morus alba) leaves. Temperature and humidity were maintained at 26 (±2) °C and 75 (±5)%, respectively, throughout the study. Observations on change in body length of larvae were made on every alternate day. We also calculated the shell ratio, time taken in cocoon formation and emergence. Results from experiment show that the larvae underwent significant change in body length but with faster growth rate under 12L:12D photoperiod in comparison to 8L:16D and 16L:8D. Time taken in cocoon formation and hatching was significantly less, while shell ratio was significantly higher in 12L:12D in comparison to other groups. We did not observe much effect of monochromatic light on the development of silkmoth.     

Olfactory responses recorded from sensilla coeloconica of the silkmoth Bombyx mori

Abstract. Extracellular single sensillum recordings were made from the double-walled multiporous sensilla coeloconica on the antennae of males and females of Bombyx mori L. (Lepidoptera: Bombycidae). The receptor neurones responded to olfactory stimuli; no thermo- or hygroreceptors were found. Many neurones responded with a decrease of the transepithelial potential and increased rates of nerve impulses to aliphatic hydrocarbons with chain lengths between three and ten carbon atoms. Most abundant were neurones responding best to acids and aldehydes. Receptor neurones responding with a low threshold to only one acid of a certain chain length were considered as specialists. In some sensilla an increase of the transepithelial potential combined with nerve impulse inhibition was observed in response to monoterpene alcohols. Neurones responding with excitation to aliphatic acids and with inhibition to terpenes were found in the same sensilla. Some neurones excited by aliphatic acids were inhibited by terpenes. Responses to headspace volatiles of mulberry leaves, the larval food, were also obtained. Therefore the coeloconic sensilla may be involved in the selection of oviposition sites.     

The Drosophila caspases Strica and Dronc function redundantly in programmed cell death during oogenesis

Programmed cell death (PCD) in the Drosophila ovary occurs either during mid-oogenesis, resulting in degeneration of the entire egg chamber or during late oogenesis, to facilitate the development of the oocyte. PCD during oogenesis is regulated by mechanisms different from those that control cell death in other Drosophila tissues. We have analyzed the role of caspases in PCD of the female germline by examining caspase mutants and overexpressing caspase inhibitors. Imprecise P-element excision was used to generate mutants of the initiator caspase strica. While null mutants of strica or another initiator caspase, dronc, display no ovary phenotype, we find that strica exhibits redundancy with dronc, during both mid- and late oogenesis. Ovaries of double mutants contain defective mid-stage egg chambers similar to those reported previously in dcp-1 mutants, and mature egg chambers with persisting nurse cell nuclei. In addition, the effector caspases drice and dcp-1 also display redundant functions during late oogenesis, resulting in persisting nurse cell nuclei. These findings indicate that caspases are required for nurse cell death during mid-oogenesis, and participate in developmental nurse cell death during late oogenesis. This reveals a novel pathway of cell death in the ovary that utilizes strica, dronc, dcp-1 and drice, and importantly illustrates strong redundancy among the caspases.     

Post-eclosion diuresis in a flightless insect, the silkmoth Bombyx mori

Abstract. Although the silkmoth, Bombyx mori L., has lost the ability to fly, it has retained a post-eclosion diuresis. In moths removed from their cocoons before eclosion, or in those which failed to spin cocoons as larvae, the weight loss due to diuresis was 14% of the eclosion body weight in males. Moths which used labial fluid to escape from their cocoons showed a correspondingly smaller diuresis (5%). Both urine and labial fluid had high potassium and low sodium concentrations. Unlike post-eclosion diuresis in butterflies, however, the urine was isosmotic to the haemolymph. In vitro preparations of B.mori Malpighian tubules were stimulated by cyclic AMP, B.mori brain extracts and Manduca sexta diuretic peptide (Mas-DP I).     

Fenoxycarb modulates ecdysone receptor B1 and programmed cell death of the anterior silk gland of silkworm Bombyx mori

Fenoxycarb, O‐ethyl N‐(2‐(4‐phenoxyphenoxy)‐ethyl) carbamate has been shown to be one of the most potent juvenile hormone analogues against a variety of insect species. In the present study, topical application of fenoxycarb to fifth‐instar larvae of the silkworm Bombyx mori (Lepidoptera: Bombycidae) was performed immediately after the fourth ecdysis (on day 0), day 3 and day 6 of the instar and then its effects on the anterior silk glands (ASG) and ecdysone receptor B1 (EcR‐B1) protein were investigated during larval pupal development. Fenoxycarb application increased the instar length and prevented metamorphic events, depending on the application time. The ASGs of B. mori undergo programmed cell death during the larval–pupal metamorphosis and an insect steroid, 20‐hydroxyecdysone (20E), triggers this cell death. The exact mechanism by which 20E and juvenile hormone regulates programmed cell death in insect tissues is poorly understood. To gain insights into how juvenile hormone regulates metamorphic events like programmed cell death in the anterior silk glands, we analyzed the progression of programmed cell death with morphological observations and biochemical experiments like acid phosphatase activity and DNA electrophoresis. Then we examined the EcR‐B1 protein levels and their relationships with programmed cell death. Our results indicated that fenoxycarb modulates programmed cell death of the anterior silk glands and EcR‐B1 protein level, depending on the application time. Fenoxycarb may exhibit its effects in at least two different ways: (i) acting on prothoracic gland secretory activity; and/or (ii) regulation of EcR‐B1 expression in the anterior silk glands for programmed cell death process.     

Fuel mobilization for energy metabolism in the silkmoth, Bombyx mori, during reproductive processes

Flight muscles of male moth, B. mori seem to utilize carbohydrate preferentially as a source of energy for all its acrobatic movements during the search for female moth. Depletion of triacylglycerol from flight muscles without affecting its level from fat body suggests that this lipid fraction serves as a source of energy in flight muscles during insemination processes. Significant depletion of triacylglycerol and glycogen from flight muscles of female moth after egg laying indicates that they are used to meet the energy requirement of female during oviposition activity. Depletion of proteins from flight muscles of male and female insects suggest that these proteins are transported to the accessory reproductive glands to meet their protein demand.     

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.     

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.     

Developmental profile of annexin IX and its possible role in programmed cell death of the Bombyx mori anterior silk gland

During pupal metamorphosis, the anterior silk gland (ASG) of the silkworm, Bombyx mori, undergoes programmed cell death (PCD), which is triggered by 20-hydroxyecdysone (20E). Annexin IX (ANX IX) has been identified as a 20E-inducible gene in dying ASGs, and we show here that its expression is down-regulated in tissues destined to die but not in tissues that survive pupal metamorphosis. ANX IX expression was high in the ASGs during the feeding period, when the ecdysteroid titer was low, and decreased in response to the rising ecdysteroid titer that triggered pupal metamorphosis. Before gut purge, in vitro exposure of the ASGs to 20E levels corresponding to the ecdysteroid concentration present at the time of gut purge caused a decrease in ANX IX messenger RNA levels. Expression profiles of EcR and USP, and the 20E concentration-responses of these genes, indicate the importance of the relative abundance of EcR-A and EcR-B1 isoforms in ANX IX regulation. These results suggest an involvement of ANX IX in the determination of PCD timing by delaying or suppressing the response to the increase in hemolymph ecdysteroid concentration during the prepupal period.     

Partial purification of prostaglandin synthetase from silkmoth Bombyx mori L.

Prostaglandin synthetase has been isolated and purified from the post mitochondrial supernatant of the testis of male silkmoth Bombyx mori L. A 3-fold purification was achieved compared to that of post mitochondrial supernatant. The partially purified enzyme converted arachidonic acid to PGE₂in vitro in the presence of haemin and hydroquinone.     

Prostaglandin signaling and ovarian follicle development in the silkmoth, Bombyx mori

Previous work on in vitro culturing of silkmoth (Bombyx mori) ovarian follicles has shown that starting from middle vitellogenesis, follicles develop according to an endogenous developmental program that does not require the presence of extra-ovarian factors. In this paper, we are reporting on our investigation for a possible involvement of autocrine/paracrine signaling by prostaglandins in the control of silkmoth ovarian follicle development. Using an initial rapid test that evaluates the formation of a protective eggshell around the oocyte, we are showing that aspirin and indomethacin, potent inhibitors of prostaglandin biosynthesis, block the transition of cultured vitellogenic follicles into choriogenesis. More detailed studies involving analyses of temporal expression patterns of genes known to be expressed in follicular epithelium cells at specific stages of ovarian development revealed that inhibition of prostaglandin biosynthesis arrests stages of follicle development from middle vitellogenesis to late choriogenesis. The arrest could be reversed by the addition of exogenous prostaglandins or cAMP into the culture media leading to the conclusion that the production of prostaglandins triggers cAMP-mediated intracellular signaling that allows the developmental progression of the follicles. Finally, because neither prostaglandins nor cAMP is capable of rescuing a developmental block effected at mid-vitellogenesis by the ecdysone agonist tebufenozide, we are proposing that prostaglandins have a role in the maintenance of normal physiological homeostasis in the ovarian follicles rather than a more specific role in developmental decision-making at distinct stages of follicle development.