Day Of Pupal Development Research Articles

Extensive transcriptomic profiling of pupal diapause in a butterfly reveals a dynamic phenotype.

Diapause is a common adaptation for overwintering in insects that is characterized by arrested development and increased tolerance to stress and cold. While the expression of specific candidate genes during diapause have been investigated, there is no general understanding of the dynamics of the transcriptional landscape as a whole during the extended diapause phenotype. Such a detailed temporal insight is important as diapause is a vital aspect of life cycle timing. Here, we performed a time-course experiment using RNA-Seq on the head and abdomen in the butterfly Pieris napi. In both body parts, comparing diapausing and nondiapausing siblings, differentially expressed genes are detected from the first day of pupal development and onwards, varying dramatically across these formative stages. During diapause there are strong gene expression dynamics present, revealing a preprogrammed transcriptional landscape that is active during the winter. Different biological processes appear to be active in the two body parts. Finally, adults emerging from either the direct or diapause pathways do not show large transcriptomic differences, suggesting the adult phenotype is strongly canalized.

Curly Encodes Dual Oxidase, Which Acts with Heme Peroxidase Curly Su to Shape the Adult Drosophila Wing.

Curly, described almost a century ago, is one of the most frequently used markers in Drosophila genetics. Despite this the molecular identity of Curly has remained obscure. Here we show that Curly mutations arise in the gene dual oxidase (duox), which encodes a reactive oxygen species (ROS) generating NADPH oxidase. Using Curly mutations and RNA interference (RNAi), we demonstrate that Duox autonomously stabilizes the wing on the last day of pupal development. Through genetic suppression studies, we identify a novel heme peroxidase, Curly Su (Cysu) that acts with Duox to form the wing. Ultrastructural analysis suggests that Duox and Cysu are required in the wing to bond and adhere the dorsal and ventral cuticle surfaces during its maturation. In Drosophila, Duox is best known for its role in the killing of pathogens by generating bactericidal ROS. Our work adds to a growing number of studies suggesting that Duox’s primary function is more structural, helping to form extracellular and cuticle structures in conjunction with peroxidases.

A proteomic view on the developmental transfer of homologous 30 kDa lipoproteins from peripheral fat body to perivisceral fat body via hemolymph in silkworm, Bombyx mori

BackgroundA group of abundant proteins of ~30 kDa is synthesized in silkworm larval peripheral fat body (PPFB) tissues and transported into the open circulatory system (hemolymph) in a time-depended fashion to be eventually stored as granules in the pupal perivisceral fat body (PVFB) tissues for adult development during the non-feeding stage. These proteins have been shown to act anti-apoptotic besides being assigned roles in embryogenesis and defense. However, detailed protein structural information for individual PPFB and PVFB tissues during larval and pupal developmental stages is still missing. Gel electrophoresis and chromatography were used to separate the 30 kDa proteins from both PPFB and PVFB as well as hemolymph total proteomes. Mass spectrometry (MS) was employed to elucidate individual protein sequences. Furthermore, 30 kDa proteins were purified and biochemically characterized.ResultsOne- and two-dimensional gel electrophoresis (1/2D-PAGE) was used to visualize the relative changes of abundance of the 30 kDa proteins in PPFB and PVFB as well as hemolymph from day 1 of V instar larval stage to day 6 of pupal stage. Their concentrations were markedly increased in hemolymph and PVFB up to the first two days of pupal development and these proteins were consumed during development of the adult insect. Typically, three protein bands were observed (~29, 30, 31 kDa) in 1D-PAGE, which were subjected to MS-based protein identification along with spots excised from 2D-gels run for those proteomes. Gas phase fragmentation was used to generate peptide sequence information, which was matched to the available nucleotide data pool of more than ten highly homologous insect 30 kDa lipoproteins. Phylogenetic and similarity analyses of those sequences were performed to assist in the assignment of experimentally identified peptides to known sequences. Lipoproteins LP1 to LP5 and L301/302 could be matched to peptides extracted from all bands suggesting the presence of full length and truncated or modified protein forms in all of them. The individual variants could not be easily separated by classical means of purification such as 2D-PAGE because of their high similarity. They even seemed to aggregate as was indicated by native gel electrophoresis. Multistep chromatographic procedures eventually allowed purification of an LP3-like protein. The protein responded to lipoprotein-specific staining.ConclusionsIn B. mori larvae and pupae, 30 kDa lipoproteins LP1 to LP5 and L301/302 were detected in PPFB and PVFB tissue as well as in hemolymph. The concentration of these proteins changed progressively during development from their synthesis in PPFB, transport in hemolymph to storage in PVFB. While the 30 kDa proteins could be reproducibly separated in three bands electrophoretically, the exact nature of the individual protein forms present in those bands remained partially ambiguous. The amino acid sequences of all known 30 kDa proteins showed very high homology. High-resolution separation techniques will be necessary before MS and other structural analysis can shed more light on the complexity of the 30 kDa subproteome in B. mori. A first attempt to that end allowed isolation of a B. mori LP3-like protein, the complete structure, properties and function of which will now be elucidated in detail.

The effect of a static magnetic field on the morphometric characteristics of neurosecretory neurons and corpora allata in the pupae of yellow mealworm Tenebrio molitor (Tenebrionidae)

Purpose: The morphometric characteristics of A1 and A2 protocerebral neurosecretory neurons (cell and nuclei size, number of nucleoli in the nuclei); corpora allata size, nuclei size, cell number, were investigated in the pupae of yellow mealworm, Tenebrio molitor (L.), exposed to a strong static magnetic field of 320 mT maximum induction (10,000 times higher than the Earth's).Materials and methods: The experimental groups of Tenebrio molitor pupae were: A control group exposed only to natural magnetic field and sacrificed at the eighth day of pupal development (C); and pupae kept in a strong static magnetic field for eight days and then sacrificed (MF). Serial brain cross-sections were stained using the Alcian Blue Floxin technique. All the parameters were analyzed and measurements were performed using an image processing and analysis system (Leica, Cambridge, UK) linked to a Leica DMLB light microscope (program is QWin – Leica's Quantimet Windows-based image analysis tool kit).Results: The values of morphometric parameters of neurosecretory neurons and corpora allata were significantly increased after exposure of the pupae to the strong magnetic field.Conclusions: The strong magnetic field influence characteristics of protocerebral neurosecretory neurons and corpora allata in the late Tenebrio molitor pupae.

Proliferation and programmed cell death of neuronal precursors in the mushroom bodies of the honeybee

We have studied proliferation and programmed cell death in the brain of the honeybee during metamorphosis. DNA fragmentation detection using the TUNEL method combined with 5-bromodeoxyuridine incorporation experiments reveal that in the mushroom bodies neurogenesis is terminated by extensive apoptosis. Proliferation of mushroom body neuroblasts is active until the fourth day of pupal development, ceasing abruptly within 1 day after the onset of apoptosis in the mushroom body proliferative clusters. Inside the mushroom bodies, apoptosis spreads from the apical ends of proliferative clusters, beneath the brain's surface, toward the basal ones. The distributions of apoptotic cells and those in the S phase of the cell cycle overlap significantly. Electron microscopic analysis gives further evidence that mushroom body neuroblasts themselves undergo programmed cell death. We suggest that programmed cell death may be the main factor controlling the final number of Kenyon cells produced during metamorphosis. The overlap in time and space between proliferation and apoptosis raises the question of whether the neuronal precursors switch to programmed cell death during the progression of the cell cycle, or afterwards.

Early retinal development in Drosophila.

Like other insects, the fruit fly Drosophila melanogaster has a compound eye, made up of approximately 800 individual eyes or ommatidia. The eye arises from the eye imaginal disk, which is part of the compound eye-antennal disk. The cells that will form this disk invaginate from the ectoderm during late embryogenesis (Jurgens and Hartenstein 1993); the disk primordium then grows inside the larva, where it consists of an epithelial bilayer. One columnar layer will give rise to all the cell types of the retina; it is covered by a squamous epithelial sheet called the peripodial membrane, which later contributes to the surface of the head (Haynie and Bryant 1986). Differentiation of the retina begins in the third of the three larval stages, known as instars, and continues during the first few days of pupal development (Ready et al. 1976; Fig. 1). In spite of the many morphological and developmental differences between fly and vertebrate eyes, many of the molecules involved appear to be conserved.KeywordsImaginal DiskMorphogenetic FurrowPhotoreceptor DifferentiationPeripodial MembraneEarly Retinal DevelopmentThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Dendritic pattern development of the honeybee antennal lobe neurons: a laser scanning confocal microscopic study.

The processing of odorant signals is performed, in the olfactory bulb of vertebrates or in the antennal lobe of insects, by different types of neurons which display specific morphological and functional features. The present work characterizes the morphogenesis of the main neuronal types which participate in olfactory discrimination in the adult honeybee (Apis mellifera). Neurons were stained intracellularly with Lucifer yellow at different stages of pupal development and in the adult, and imaged by laser scanning confocal microscopy. Attending to branching patterns, all pupal neurons could be attributed to morphological types previously established in the adult. Given the functional importance of intraglomerular dendritic arbors in the processing of olfactory information, the study focused on their development. The two main classes, dense and sparse intraglomerular arbors, display adultlike features as early as the second day of pupal development. However, morphometric measurements and confocal observations show that their general pattern undergoes continuous maturation processes until late pupal stages and after emergence of the adult. Among these, the results point out a pruning of dendritic branches in sparse arbors, but not in dense arbors.

The pleiotropic function of Delta during postembryonic development of Drosophila melanogaster.

Analysis of the development of Delta (Dl) temperature-sensitive mutants pulsed at restrictive temperature during larval and pupal stages reveals multiple phenocritical periods during which reduction of Dl function affects viability and development of adult structures. Dl function is required during the third larval instar for post-pupal viability and during the first day of pupal development for viability through eclosion. Dl function is required biphasically for the development of sensory bristles. Earlier pulses lead to bristle multiplication and later pulses lead to bristle loss. The exact intervals during which multiplication and loss are induced vary for different bristles. Dl function is also required for development of most, if not all, cell types in the retina. Different pulses result in reduction in eye size, scarring, and glossiness, as well as multiplication and loss of interommatidial bristles. We also define intervals during which Dl function is required for aspects of leg and wing development. Phenocritical periods for Dl function are temporally coincident with those previously reported for Notch (N), consistent with the hypothesis that the proteins encoded by Dl and N interact throughout development to assure correct specification of cell fates in a variety of imaginal tissues.

MRNAs of wing epidermis of the oak silkmoth Antheraea polyphemus and other silkmoths during pupal development

The mRNA populations of wing epidermal cells of the silkmoth Antheraea polyphemus during juvenile hormone induced second pupal development have been analyzed by two techniques. The analysis of the data derived from hybridization of cDNAs to the corresponding mRNAs demonstrate that the mRNAs are complex during the entire period of pupal development. Each mRNA population hybridizes to its cDNA over five orders of Rot and belong to three abundance classes. A comparison of the hybridization of two mRNAs derived from the wing epidermis at the beginning of development and at the end of pupal development with each other's cDNAs shows that only about 60% of the messages are common between the two stages. The 40% of the mRNAs that are new and abundant at the time of second pupal cuticle synthesis have been isolated as pupal specific (null) cDNAs and characterized by hybridization to various mRNAs. Quantitative analysis of the data shows that the mRNAs corresponding to these specific cDNAs are about 200 in number and appear at 4 days of pupal development. The mRNAs derived from the pupal wing epidermis of three other silkmoths can also hybridize to the polyphemus pupal specific cDNAs to varying extents demonstrating the presence of similar mRNAs in their wing epidermis at the appropriate time. The differences in the hybridization patterns seen with the various mRNA populations and the appearance of new and abundant mRNAs at 4 days of pupal development have been correlated with two dimensional gel electrophoretic patterns of the in vitro translation products from the same mRNAs. A comparison of these patterns demonstrate the appearance of several new mRNAs at the time of pupal cuticle synthesis. The translation products of these mRNAs appear to have isoelectric points and molecular weights similar to those of the major pupal cuticular polypeptides. The translation products of the mRNAs derived from the pupal wing epidermis of other silkmoths also yield 2-D gel patterns quite similar to that of polyphemus silkmoth. The hybridization data and the 2-D gel patterns of the translation products of mRNAs isolated from the abdominal and pupal wing epidermis derived from primary pupae are indistinguishable from those of mRNAs derived from secondary pupal wing epidermis.

The genetic control of cell proliferation in Drosophila imaginal discs

The imaginal discs of Drosophila provide a favorable system for the analysis of the mechanisms controlling developmental cell proliferation, because of the separation in time between cell proliferation and differentiation, and the facility with which controlling genes can be identified and characterized. Imaginal discs are established in the embryo, and grow by cell proliferation throughout the larval period. Proliferation terminates in a regular spatial pattern during the final stages of larval development and the first day of pupal development. Cell proliferation can be locally reactivated in growth-terminated imaginal discs by removing part of the disc and culturing the remaining fragment in an adult host. The pattern of proliferation in these fragments suggests that cell proliferation in imaginal discs is controlled by direct interactions between cells and their neighbors. Proliferation appears to be stimulated by positional information differences, and these differences are reduced by the addition of new cells during tissue growth. Genes involved in cell proliferation control have been identified by collecting and analyzing recessive lethal mutations which cause overgrowth of imaginal discs. In some of these mutants (fat, lgd, c43, dco) the overgrowing tissue is hyperplastic; it retains its single-layered epithelial structure and is capable of differentiating. In two of the hyperplastic mutants (dco and c43), the imaginal discs show a failure of gap-junctional cell communication, suggesting that this form of cell communication may be involved in termination of proliferation. In other mutants the overgrowing disc tissue is neoplastic: it loses its structure and ability to differentiate, becoming a tumorous growth. The two genes that give a neoplastic phenotype (dlg and lgl) have been cloned and cDNAs of one of them (lgl) sequenced. The lgl gene encodes a cell surface molecule with significant homology to calcium-dependent cell adhesion molecules (cadherins). The expression of lgl at the time of termination of cell proliferation suggests that there are changes in the way that cells interact with one another at these times, and that these changes may be implemented by cell adhesion molecules. Direct cell contact within the epithelium, as well as signalling through gap junctions, appears to be involved in the cell interactions needed for the termination of cell proliferation. Mutations in genes encoding the Drosophila homologs of growth factors, growth factor receptors and oncogenes usually show an effect on cell-fate decisions rather than cell proliferation control, but this may be because oncogenic mutations in these genes would be dominant lethals and would therefore not be identified by conventional genetic analysis.

A variant β-tubulin isoform of Drosophila melanogaster (β3) is expressed primarily in tissues of mesodermal origin in embryos and pupae, and is utilized in populations of transient microtubules

The beta 3-tubulin gene of Drosophila melanogaster codes for a variant tubulin isoform which is expressed at two distinct times during development: (1) during midembryogenesis from 8-16 hr postfertilization, and (2) during the 4 days of pupal development. We have determined the spatial pattern of beta 3-tubulin expression by localizing the beta 3 mRNA in paraffin sections using a 3' message-specific RNA probe and by localizing the beta 3 protein using a polyclonal antibody specific for Drosophila beta 3-tubulin. During embryogenesis beta 3 is restricted to and is expressed in all of the developing muscles. During pupal development beta 3 is also expressed at high levels in developing adult muscles. In addition, early in pupal development beta 3 is expressed in the imaginal discs, while at later times beta 3 is expressed in the epidermal cells of the wing blade, the optic lobe, the ovaries, and the testes. The expression of beta 3 tubulin ceases by the end of pupal development in all of these tissues except the ovaries and testes where expression persists into the adult. In both developing muscles and wings our results indicate that beta 3-tubulin is utilized in populations of specialized but transient cytoskeletal microtubules which are involved in establishing the final form of the tissue.

Irreversible Damage to Flight Muscles Resulting from Disturbance of Pupae during Rearing of the Mediterranean Fruit Fly, Ceratitis capitata1

The agitation involved in the sifting of pupae of Mediterranean fruit flies, Ceratitis capitata (Wiedemann), from the substrate in which they have pupated results in an irreversible effect on the flight muscles if the sifting occurs during the 4th through 6th days of pupal development at 21°C. The effect consists of the separation of the attachment of the median dorsal muscles from the posterior phragma, so that the affected muscles are attached only at their anterior ends. This detachment, and the resulting shortening of the muscles, was found to have occurred even in pupae sampled immediately after the sifting process. During the remaining days of pupal development, the affected muscles remain only as much-shortened stubs attached to, and lying close against, the anterior, dorsal thoracic body wall. Adult flies from affected pupae cannot fly.

Analysis of a newly‐isolated temperature sensitive maternal effect mutation of Drosophila melanogaster

A mutation located near the tip of the X chromosome in Drosophila melanogaster has been isolated, and its developmental effects described. This mutation (1(1)ts-1 is temperature sensitive, and at permissive temperature (18 degrees C) develops normally. However, zygotes from females raised or aged at restrictive temperature (28 degrees C) never hatch, regardless of the embryonic genotype. Midgut formation is abnormal in lethal zygotes and dorsal closure is probably incomplete. Temperature shift experiments have shown that the zygotic lethality is governed by a temperature sensitive period in oocytes of stage seven or older. If viable 1(1)ts-1 embryos are shifted to restrictive temperatures, they develop as far as the pupal stage, but never eclose. The temperature sensitive period for pupal lethality includes the last 2.5 days of pupal development and does not involve a maternal effect.

BIOSYNTHESIS OF ECDYSONE IN THE ISOLATED ABDOMEN OF THE SILKWORM, BOMBYX MORI.

During pupal-adult development of the silkworm, Bombyx mori, the ecdysone titer changes, exhibiting two maxima in the females: one on the second day of pupal development and the other just before adult emergence. During the second maximum, ecdysone accumulates in the ovaries. It also accumulates in isolated abdomens, which were prepared just after pupal ecdysis and induced to initiate adult development by injection with β-ecdysone. Several lines of evidence suggest that ecdysone is synthesized in the ovary itself.

ANALYSIS OF SOME TEMPERATURE EFFECTS ON DROSOPHILA PUPAE

1. A previously presented scheme unifying a variety of high temperature effects on day-old Drosophila melanogaster pupae has been confirmed substantially by a large body of new data. Several minor modifications have been made. 2. New experiments confirm the validity of analyzing heat-induced temperature adaptation over a several-hour period at the end of the first day of pupal development. 3. Several new types of experiments have been described which involve combinations of exposures to more than one temperature and which provide information on the transitions between several intermediate stages in the scheme. 4. A double temperature-dependence is demonstrated in which both the amount of a precursor and the rate of its conversion vary with temperature in a particular range. In this range, the temperature coefficient of crossvein defect induction is the product of two components. This property inheres in branched pathways. 5. Rate constants and temperature coefficients have been calculated for the individual steps on the basis of data from specific experiments. 6. The scheme incorporates the information from a large array of detailed experiments and is capable of generalization over a broad temperature range.

The water balance of tsetse pupae

1. The pupal period of Glossina morsitans can be divided into a number of different stages on the basis of anatomical changes: (a) The 3rd instar larva which comprises the first day of pupal development. During this time water is lost at relatively high rates in part by transpiration through the puparium and in part through the supernumerary stigmata of the polypneustic lobes. (b) The 4th instar larva which comprises the next 3 days; rates of transpiration are somewhat lower due to the deposition inside the puparium of a second integument. But water is lost by excretion as well as by transpiration; this excretory loss is regulated so that the more water is lost by (c) The pupa and pharate adult. The larva-pupa moult occurs on about the 5th day and a few days later the hypodermis retracts from the pupal skin and the adult cuticle begins to be laid down. Following the formation of the very impermeable pupal skin there is a substantial decrease in the rate of transpiration. At this stage water is lost also through the pupal spiracle, but this loss is under active control, the rate being a function of the water content of the pupa. (d) A few days before emergence the pupal skin dries out and its permeability increases ; spiracular control ceases at the same time, so that rates of water loss during this period are high . 2. The regulation of excretory loss and of spiracular loss, combined with extremely low membrane permeabilities, confers a high degree of resistance to desiccation on the pupa of ; it can complete its development at humidities as low as 10 /0 r.h. 3. It is shown that in areas where a prolonged dry season occurs the humidity to which pupae are subject under natural conditions may fall to about 40 % r.h.; if the pupal sites are exposed to the action of grass fibres even lower humidities may be experienced. 4. There is a close correlation between the resistance to desiccation of the pupae of different species of tsetse fly and the habitat in which they occur, and it is suggested that the water balance of the pupa may have been an important limiting factor in the invasion of semi-arid and arid habitats. 5. The development of resistance to desiccation in tsetse pupae has involved a decrease in the permeability of puparium and pupal skin, a decrease in the size of respiratory lobes and an increase in the percentage loss of water which can be tolerated before development stops.