Early Pupal Stage Research Articles

Twist Is Required for Muscle Template Splitting during AdultDrosophilaMyogenesis

The basic helix–loop–helix transcription factor Twist is required for normal development of larval and adult somatic muscles inDrosophila.Adult flies normally have six pairs of dorsal longitudinal indirect flight muscles (DLMs), whereas when Twist function is reduced, only three pairs of DLMs are formed. Althoughtwistis expressed in precursors of adult muscles throughout the larval and early pupal stages, we demonstrate that Twist function is required only during the late larval stage for DLM patterning. In wild-type flies, this is just prior to the time when three pairs of persistent larval muscle fibers split longitudinally to form templates for the six pairs of DLMs. By examining sections at various times during pupal development, we found that splitting of the larval muscles does not occur intwistmutants, indicating that Twist function is required to induce major changes in the larval templates prior to differentiation. The function of Twist in larval muscle splitting is likely mediated by myocyte enhancer factor-2 (MEF2) since inMef2hypomorphic mutants splitting is also reduced andMef2expression is dependent upon Twist. Our findings define specific roles for Twist and MEF2 during pupal myogenesis and demonstrate that these transcription factors function in adult muscle precursor cells to regulate downstream factors controlling muscle cell splitting and morphogenesis.

Development of the giant fiber neuron ofDrosophila melanogaster

The giant fiber system (GFS) of Drosophila melanogaster provides a convenient system in which to study neural development. It mediates escape behaviour through a small number of neurons, including the giant fibers (GFs), to innervate the tergotrochantral jump muscle (TTM) and the dorsal longitudinal flight muscles. The GFS has been intensively studied physiologically in both wild-type and mutant flies, and is often used as a system to study the effects of neural mutations on the physiology of the adult nervous system. Recently, much information has been gleaned as to how and when synaptogenesis, with its major target neurons, is achieved. However, little is known of the earlier development of this neuron. Here we have used an enhancer-trap, marking parts of the GFS, in conjunction with BrdU labelling, to attempt to follow the birth, axonogenesis, and the early morphological meeting of the GFs with their target neurons. From these anatomical observations we propose that the GF cell is not born during the larval or pupal stages and, therefore. appears to be a persistent embryonic cell. The axons of the GFs develop during the third instar. During the early pupal stages the GFs contact other identified neurons of the GFS. In addition, we see some aberrant development of the network, with some flies carrying only one GF, and yet others with extended axons. We present a model for the initial joining of the GFs and tergotrochanteral motorneurons (TTMns).

Molecular characterization of the Notch homologue from the Australian sheep blowfly, Lucilia cuprina

The Drosophila melanogaster Notch gene product functions as a receptor of intercellular signals and is central to cell fate specification. The Scalloped wings ( Scl) gene is the homologue of Notch in the Australian sheep blowfly, Lucilia cuprina. An allele of Scl is thought to be involved in the modification of Darwinian fitness and bristle asymmetry in flies resistant to organophosphorous chemicals (OPs). As a first step towards the testing of this hypothesis we cloned and sequenced Scl. A full-length cDNA segment representing the mRNA of Scl is 8503 bp and encodes a protein of 2653 amino acids, which shares 73.6% identity with Notch. All functional motifs including EGF-like repeats, LNR repeats, cdc10/ankyrin repeats, opa and PEST elements are present in the same order as in Notch and the sequence identities peak in these motifs. With respect to genomic structure, intron/exon boundaries are conserved but, in most cases, the Scl introns are larger. Sequence analysis of the upstream genomic region reveals that the gene has a TATA-less promoter. Consistent with a central role in embryogenesis and imaginal development, high levels of Scl expression were detected in the early embryonic and pupal stages.

Identification and developmental profiles of hexamerins in antenna and hemolymph of the honeybee, Apis mellifera

Four distinct hexamerin subunits (referred to as « hexamerins » in the following text) have been identified in the developing honeybee, Apis mellifera, by N-terminal protein sequencing. Hexamerins are abundant in the hemolymph of late larval and early pupal stages, and gradually decline during metamorphosis and adult development. Three hexamerins in the 70 kDa range have been found (Hex70a, Hex70b, Hex70c). In worker and drone, Hex70a is the only hexamerin present in large amount in later adult stages. Hex70b and c exhibit a similar developmental profile, disappearing in the drone just before adult emergence, and in the worker just after. Hex70b or Hex70c are still detectable in the adult queen. Hex80/110 likely exist in at least 3 different subunits, 1 of 110 kDa, and 2 of around 80 kDa, which all share a common N-terminus. They disappear during metamorphosis earlier than Hex70b and c. All these hexamerins have been found also in the antenna, suggesting their utilization in building up of antennal cuticle structures.

Insect larvae contain substances toxic to adults: the discovery of paralysins

Acidic methanolic extracts of larvae of nine different insect species were found to contain substances that cause a lethal effect in the adult stage of the same species and of other species. These endogenous toxic substances, apparently being widely spread over the class of insects, were designated as paralysins, because of their immediate and observable paralytic effect upon injection. The developmental concentration curves of five different species of insects ( Galleria mellonella (Lepidoptera), Neobellieria bullata (Diptera), Spodoptera frugiperda (Lepidoptera), Tenebrio molitor (Coleoptera) and Schistocerca gregaria (Orthoptera) indicate that the toxins are not present throughout all the developmental stages in the same concentration. The strongest paralytic activity was found in late instar larvae or in the early pupal stage. The temporal distribution of paralysins during development suggests that they might be involved in metamorphosis.

Establishment of stem cell identity in the Drosophila germline.

In the adult Drosophila ovary the continuous production of eggs depends upon a small group of stem cells located at the anterior tip of the germarium. These stem cells divide asymmetrically to self renew and to generate a cystoblast, which in females is committed to the oocyte differentiation pathway. While much is known about the development of poststem cell cystoblasts, little is known about when stem cells are formed or how their identity is initially established. To investigate these questions we have used the P-M hybrid dysgenesis syndrome as a tool for ablating the "pre-stem cell" progenitors of the stem cells. Our experiments indicate that the pre-stem cells in females assume stem cell identity during the early pupal stage. Our results also suggest a model in which at least two pre-stem cells must be present within an ovariole for the specification of stem cell fate. When only a single pre-stem cell is sequestered by an ovariole, this cell does not assume stem cell identity, but instead follows the cystoblast-cystocyte differentiation pathway.

Development changes in juvenile hormone and juvenile hormone acid titers in the hemolymph and in-vitro juvenile hormone synthesis by corpora allata of the silkworm, Bombyx mori

A simple method was developed to quantify hemolymph juvenile hormone (JH) and JH acid in hemolymph extracts from Bombyx mori with an established radioimmunoassay (RIA) for JH I. When various organic solvent extracts of hemolymph were assayed by RIA, levels of non-specific binding of the labeled ligand in the assay were determined to be greater than 50% of the maximum amount of the label bound by the antiserum. When hemolymph was diluted with methanol:water:8.4 N ammonium hydroxide (10:9:1) and extracted with isooctane, non-specific binding was only 50% higher than control levels obtained with the assay buffer alone. The organic phase contained only JH and aqueous phase, JH acid. Consequently, this extraction method was used to prepare samples for RIA and enabled the separate measurement of JH and JH acid in hemolymph. With this method, changes in the hemolymph titers of JH and JH acid were determined from the third instar through early pupal stage of Bombyx mori. Changes in the in vitro secretory activity of corpora allata and brain–corpora cardiaca–corpora allata complexes from fifth instar larvae were also determined by using JH I RIA of the incubation medium.

Development of adult thoracic leg muscles during metamorphosis of the hawk moth Manduca sexta

During metamorphosis, the larval thoracic legs of the hawk moth Manduca sexta are replaced by a new set of adult legs. The larval leg motoneurons persist to innervate new adult muscles, and the motor terminals remain within the developing adult legs. Here we describe the fate of the larval leg muscles and the origin of new muscles within the adult legs. During the larval instars, large and small nuclei proliferate within leg muscle fibers. Near the end of the larval stage a subset of the small nuclei undergo a wave of proliferation, as indicated by the incorporation of 5-bromodeoxyuridine, whereas other nuclei die. However, none of the larval leg muscles fibers persist to serve as templates for adult muscle formation, and there was no evidence for persistence of larval myonuclei. Migrating myoblasts that are born within aggregate to form adult muscle anlagen at specific production sites within the developing imaginal legs. Intense nuclear proliferation occurs within the anlagen during the early pupal stage, followed by muscle fiber formation and striation. We conclude that adult leg muscles form mainly, if not exclusively, from migrating myoblasts that without the involvement of larval elements.

Cytogenetic mapping of the Lethal hybrid rescue gene of Drosophila simulans.

Hybrids not carrying Drosophila simulans X chromosome derived from the cross between D. melanogaster females and D. simulans males are lethal at the late third instar larval period or early pupal stage. This lethality can be rescued by the mutation Lethal hybrid rescue (Lhr) of D. simulans. The Lhr gene is considered to play an important role in reproductive isolation, but the genetic analyses have not been carried out extensively because of the lack of visible mutations and chromosome rearrangements in D. simulans. Using a cuticle mutation, jabara, of which locus is close to Lhr, and D. melanogaster deficiencies, we performed cytological mapping in hybrids and estimated the Lhr locus to 54E-F.

Changes in acetylcholinesterase during pupal development of Apis mellifera queen

Total, membrane, and soluble acetylcholinesterase (AchE, EC 3.1.1.7) activities increase during the pupal development of Apis mellifera queen to reach maximum values at emergence. Membrane and soluble AchE are inhibited by 10-5 M eserine or BW284C51 except at Pr, Pdm, and Pdd stages in which soluble AchE presents eserine-sensitive and eserine-resistant fractions. At all pupal stages, AchE occurs in a major amphiphilic membrane form that represents about 98% of total AchE activity and whose sedimentation coefficient is about 5.7S, and in a minor hydrophilic form that represents about 2% of total AchE activity and whose sedimentation coefficient is about 7S. At all pupal stages, phosphatidylinositol-specific phospholipase C (PI-PLC) and glycosyl phosphatidylinositol-specific phospholipase D (GPI-PLD) convert the membrane form into soluble counterparts which electrophoretic mobilities differ from that of the soluble form. AchE exhibits a butyrylcholinesterase (BuChE) activity that represents about 14% of AchE activity. During pupal development, the BuChE/AChE ratio of the membrane fraction is relatively stable, whereas the BuChE/AChE ratio of the soluble fraction is subjected to significant variations. At early pupal stages (Pw–Pd), membrane AchE displayed a high Km value, higher than 40 μM, that decreases to an intermediary value of about 30 μM at Pdl and Pdm stages, to reach finally about 20 μM at Pdd and emergence stages. Arch. Insect Biochem. Physiol. 36:69–84, 1997. © 1997 Wiley-Liss, Inc.

Screening Test for Insecticides Interfering with Cuticular Sclerotization

The potential of known and new insecticides to interfere with cuticle sclerotization was investigated using assays for key enzymes such as phenoloxidase, quinone methide isomerase and DOPA decarboxylase. Homogenates from the blowfly Lucilia cuprina and from the epithelial cell line from Chironomus tentans were used to examine the compounds under investigation. Phenoloxidases are known to oxidize DOPA, the substrate for DOPA decarboxylase. Since phenoloxidases were not detectable in C. tentans cell homogenates, inhibitor and kinetic studies were done for comparison with DOPA decarboxylase of this insect cell line. DOPA decarboxylase and phenoloxidase of L. cuprina exerted highest specific activities at early pupal stages (day 7). The apparent K m values for the two enzymes were 0.47(±0.21) mM and 0.71(±0.16) mM, respectively, using L-DOPA as substrate. DOPA decarboxylase from C. tentans had a K m value of 0.42(±0.18) mM. Quinone methide isomerase was most active in young pupae. In terms of substrate specificity for enzymic (mushroom-tyrosinase) production of different quinones from their corresponding catechols, that with dopamine quinone proved to be the most efficient. Synthesis of derivatives of L-DOPA and L-tyrosine led to a compound which inhibited both phenoloxidase and quinone methide isomerase. DOPA decarboxylase from L. cuprina and from cells of C. tentans was inhibited by carbidopa (IC 50 values of 0.021(±0.011) μM and 0.031(±0.019) μM, respectively) and indomethacine (IC 50 values of 22.6(±7.1) μM and 18.8(±9.7) μM). Both compounds exerted a competitive type of inhibition and were able to interfere with development of L. cuprina.

Screening Test for Insecticides Interfering with Cuticular Sclerotization

The potential of known and new insecticides to interfere with cuticle sclerotization was investigated using assays for key enzymes such as phenoloxidase, quinone methide isomerase and DOPA decarboxylase. Homogenates from the blowfly Lucilia cuprina and from the epithelial cell line from Chironomus tentans were used to examine the compounds under investigation. Phenoloxidases are known to oxidize DOPA, the substrate for DOPA decarboxylase. Since phenoloxidases were not detectable in C. tentans cell homogenates, inhibitor and kinetic studies were done for comparison with DOPA decarboxylase of this insect cell line. DOPA decarboxylase and phenoloxidase of L. cuprina exerted highest specific activities at early pupal stages (day 7). The apparent Km values for the two enzymes were 0·47(±0·21) mM and 0·71(±0·16) mM, respectively, using L-DOPA as substrate. DOPA decarboxylase from C. tentans had a Km value of 0·42(±0·18) mM. Quinone methide isomerase was most active in young pupae. In terms of substrate specificity for enzymic (mushroom-tyrosinase) production of different quinones from their corresponding catechols, that with dopamine quinone proved to be the most efficient. Synthesis of derivatives of L-DOPA and L-tyrosine led to a compound which inhibited both phenoloxidase and quinone methide isomerase. DOPA decarboxylase from L. cuprina and from cells of C. tentans was inhibited by carbidopa (IC50 values of 0·021(±0·011) μM and 0·031(±0·019) μM, respectively) and indomethacine (IC50 values of 22·6(±7·1) μM and 18·8(±9·7) μM). Both compounds exerted a competitive type of inhibition and were able to interfere with development of L. cuprina.

Purification and cDNA cloning of the alcohol dehydrogenase of the flesh fly Sarcophaga peregrina. A structural relationship between alcohol dehydrogenase and a 25-kDa protein.

We purified to homogeneity two proteins with molecular masses of 25 kDa from the fat body of the Sarcophaga larva. One was alcohol dehydrogenase (ADH) and the other was a 25-kDa protein of which the genomic DNA had been cloned. We isolated the cDNA for ADH and determined its amino acid sequence. Amino acid sequence identity between ADH and the 25-kDa protein was 40%, indicating that they are structurally related proteins. The amount of ADH in Sarcophaga was almost constant through the larval stage to the adult stage, but the 25-kDa protein was detected only within a restricted period between the final larval instar and the early pupal stage.

Neural lnactivation of Sex Pheromone Production in Mated Females of the Silkworm Moth, Bombyx mori

Pheromone content in the pheromone gland of the female moth, Bombyx mori, declines after mating with a time course closely resembling that of decapitated females. The inactivation of pheromone production after mating was prevented when the ventral nerve code or a pair of peripheral nerves (N4) extending from the terminal abdominal ganglion was severed before mating. In contrast, the post-mating inactivation of pheromone production was not prevented when the ventral nerve cord was transected 1 hr after the initiation of mating. Although females produced only a small amount of pheromone when the connection between the brain and the suboesophageal ganglion (SG) was cut at an early pupal stage, mating did not induce a significant decline of pheromone production in these females. The results suggest that inactivation of pheromone production is mediated by a neural signal, originating from a peripheral receptor, that is sent via the ventral nerve cord to the brain-SG complex to suppress activity of the neurosecretory cells responsible for the release of pheromonotropic neuropeptides, such as a pheromone biosynthesis activating neuropeptide (PBAN).

124 チカイエカ蛹化前後におけるピリプロキシフェン感受性の変動

124 チカイエカ蛹化前後におけるピリプロキシフェン感受性の変動

Neurons and Ecdysteroids Promote the Proliferation of Myogenic Cells Cultured from the Developing Adult Legs ofManduca sexta

During metamorphosis in the hawkmothManduca sexta,larval leg motoneurons survive the degeneration of their target muscles to innervate new muscles that form during the development of the adult legs. Observations of muscle developmentin vivosuggest that there are close interactions between motor terminals and the muscle precursor cells at the earliest stages of muscle formation and surgical denervation compromises further development of adult muscles. Here we describe a nerve/muscle coculture system that allows further exploration of this critical developmental interaction. Muscle precursor cells derived from the developing thoracic legs of early pupae and cultured in the presence of neurons assumed a spindle-like morphology and fused to form multinucleate contractile myotubes. Contractile fibers did not form in cultures of muscle precursor cells alone. In the presence of neurons the rate of bromodeoxyuridine (BrdU) incorporation into myonuclei was significantly enhanced, suggesting that neurons promote the proliferation of myogenic cells. This effect was not unique to thoracic leg motoneurons of the early pupal stage, in that larval thoracic neurons as well as neurons from the pupal brain or abdominal ganglia were also effective at enhancing BrdU incorporation and the formation of contractile muscle fibers. Medium conditioned by neurons was ineffective at promoting BrdU incorporation, and in cocultures BrdU incorporation was enhanced only in regions of physical overlap between neurons and muscle precursor cells, suggesting that a very close-range interaction was involved. Tetrodotoxin-sensitive neuronal activity was not required for the effect on muscle development, but fixed neurons were ineffective. The insect steroid hormone 20-hydroxyecdysone enhanced BrdU incorporation into the nuclei of myogenic cells in both the presence and the absence of neurons. The results suggest that both neurons and ecdysteroids play an important regulatory role in adult muscle development, at least in part by enhancing the proliferation of myogenic cells.

DNA Synthesis of Wing Disc Cells and the Effects of Mitomycin C and X Ray Irradiation on the Wing Development of Bombyx mori

The rate of DNA synthesis of wing discs and the effects of X ray irradiation and mitomycin C injection were observed during last larval and early pupal stage of Bombyx mori. The rate of DNA synthesis in wing discs of Bombyx mori was estimated by means of the incorporation of 3H-thymidine during the fifth larval stage. Mitomycin C injection and X ray irradiation were performed during the fifth larval instar and pupal stage to investigate the effects of inhibition of DNA synthesis. The rates of 3H-thymidine incorporation were high on days 0, 1, 2, 4, 5, 7, 8 and 10 of the fifth larval instar. Wings of eclosed adults were affected by injections of mitomycin C between the 7th day of the fifth larval instar and the second day of the pupal stage. Exposing the insects to X rays during the fifth larval instar affected wing and scale formation, and at earlier stages, it had a more deleterious effect. Irradiation at 24–36 hours after pupation inhibited wing expansion and scale formation most among pupal stages. The results suggested that the peaks of 3H-thymidine incorporation during the fifth larval instar and the pupal stage have specific significance for wing development.

Suppressor of Hairless is required for signal reception during lateral inhibition in the Drosophila pupal notum.

Suppressor of Hairless (Su(H)) activity is zygotically required in larval imaginal discs for the singling out of adult sense organ precursor (SOP) cells: loss of Su(H) function results in too many proneural cluster cells adopting the SOP fate, while overexpression of the Su(H) protein prevents SOP specification. Su(H) null mutant alleles are recessive lethal at the late larval and early pupal stages. The development of Su(H) mutant cells in pupae was therefore studied in somatic clones. Clonal analysis first showed that Su(H) is required for the regular spacing of microchaete precursor cells, as clusters of mutant SOPs were detected at positions where singled out sense organ cells are normally found. Second, Su(H) mutant SOPs produced neuron-like cells, consistent with a late defect in Notch (N) signalling. Third, a careful cell-by-cell analysis of clone borders showed that Su(H) mutant cells may adopt the SOP fate even when directly adjacent to wild-type cells. Finally, quantitative clone border analysis indicates that the relative level of Su(H) gene dosage appears to bias the selection of the future SOP: cells with a higher level of Su(H) activity are more likely to adopt the epidermal fate. These results show that notum cells strictly require Su(H) activity for receiving the lateral inhibitory signal. Thus, the DNA-binding protein encoded by the Su(H) gene may act downstream of the N receptor to implement the epidermal, non-SOP fate.

NEUROANATOMICAL STUDIES OF PROTHORACIC GLANDS OF COTTON BOLLWORM HELZCOVERPA ARMZCERA (LEPIDOPTERA: NOCTUIDAE)*

Abstract Gross anatomy, ultrastructure, innervation and ultrastructural alterations of the prothoracic gland (PTG) of cotton bollworm, Helicover pa armigera (Lepidoptera: Noctuidae) are illustrated for the last larval and early pupal stages as observed by light and electron microscopy. The T‐shaped, paired (PTGs) consist each of 76–116 cells which are classified morphologically as large and small gland cells. In addition, another kind of small (about 6μ in diameter) gland cell was found in the PTGs of last instar larvae. The PTGs are innervated by the branches of 3 nerves! and tracheae and tracheoles are abundantly distributed to these glands. PTGs disappeared completely by the third day after ecdysis to the pupal stage (at temperature 28 C with a photoperiod L15:D9). An intercellular channel system (ICS) is formed by numerous, deep invaginations of the plasma membrane of gland cells. This ICS gradually increases in depth and width and reaches maximum development around the time of the major ecdysteroid secretion peak during the last larval instar.Numerous multivesicular sacs (MVS) with their remnants and an extensive rough endoplasmic reticulum were observed within ICS and cytoplasm, respectively, on the fourth day of the last larval instar. At that time the matrix of mitochondria became much more electron lucent.Freeze‐fracture replicas of the glandular epithelium were made from last instar (4th day larvae. Dynamics of structure are related to data from others concerning secretory states of the prothoracic glands of this species.

Concentration of hemolymph proteins during postembryonic worker development of Africanized honey bees in Brazil and Carniolans in Europe

The profiles of hemolymph protein content during postembryonic development of honey bees (Apis mellifera) were determined for the first time. Larval and pupal worker stages of Africanized honey bees in Brazil and Carniolans in Germany were studied. A rapid increase in protein concentration was found in the late larval stages, followed by a decrease in prepupae and a minimum amount near the pupal moult period. In the early pupal stages, the protein concentration increased again, and then decreased until adult eclosion. Only the first protein peak in spinning L5 was more pronounced in the Africanized honey bees. No differences between the hemolymph protein profiles of Africanized bees and Carniolans could be detected during pupal development. The duration of several larval and pupal stages was shorter in Africanized than in Carniolan workers. The data reflect the general characteristics of the Africanized biotype reared in tropical conditions exhibiting accelerated patterns of development and lower body weight at emergence in comparison to the European Apis subspecies reared in temperate conditions. The ontogenetic patterns described for hemolymph protein concentration in preimaginal honey bee development are also discussed, specifically in regard to the regulation of metamorphosis in holometabolous insects