The ecdysone receptor and ultraspiracle regulate the timing and progression of ovarian morphogenesis during Drosophila metamorphosis

Ecdysteroids regulate insect metamorphosis through the edysone receptor complex, a heterodimeric nuclear receptor consisting of the ecdysone receptor (EcR) and its partner ultraspiracle (USP). Differentiation in the Drosophila ovary at metamorphosis correlates with colocalization of USP and the EcR-A isoform in all but one of eight mesoderm-derived somatic cell types. The one exception is the larval terminal filament (TF) cells, in which only USP is detectable during cell differentiation. In cells destined to form the basal stalks and anterior oviduct, USP colocalizes with what appears to be the EcR-B2 isoform. Flies heterozygous for a deletion of the EcR gene exhibit several defects in ovarian morphogenesis, including a heterochronic delay in the onset of terminal filament differentiation. Flies heterozygous for a strong usp allele exhibit accelerated TF differentiation. Flies simultaneously heterozygous for both EcR and usp have additional phenotypes, including several heterochronic shifts, delayed initiation and completion of terminal filament morphogenesis and delayed ovarian differentiation during the first day of metamorphosis. Terminal filament morphogenesis is severely disrupted in homozygous usp clones. Our results demonstrate that proper expression of the ecdysone receptor complex is required to maintain the normal progression and timing of the events of ovarian differentiation in Drosophila. These findings are discussed in the context of a developmental and evolutionary role for the ecdysone receptor complex in regulating the timing of ovarian differentiation in dipteran insects.     

Severe pulmonary hemorrhage in the premature newborn infant: analysis of presurfactant and surfactant eras

Insect molting and metamorphosis are orchestrated by ecdysteroids with juvenile hormone (JH) preventing the actions of ecdysteroids necessary for metamorphosis. During the molt and metamorphosis of the dorsal abdominal epidermis of the tobacco hornworm, Manduca sexta, the isoforms involved in the ecdysone receptor (EcR)/Ultraspiracle (USP) complex change with the most dramatic switch being the loss of USP-1 and the appearance of USP-2 during the larval and pupal molts. We show here that this switch in USP isoforms is mediated by high 20-hydroxyecdysone (20E) and that the presence of JH is necessary for the down-regulation of USP-1 mRNA. The decrease of USP-1 mRNA in day 2 fourth instar larval epidermis in vitro required exposure to a high concentration (10(-5) M) of 20E equivalent to the peak ecdysteroid concentration in vivo, whereas the increase of USP-2 mRNA occurred at lower concentrations (effective concentrations, EC50=6.3x10(-7) M). During the pupal molt of allatectomized larvae which lack JH, USP-2 mRNA increased normally with the increasing ecdysteroid titer, whereas USP-1 mRNA remained high until pupation. When day 2 fifth instar larval epidermis was exposed to 500 ng/ml 20E in the absence of JH to cause pupal commitment of the cells by 24 h, USP-1 RNA remained at its high preculture level for 12 h, then increased two- to threefold by 24 h. The increase was prevented by the presence of 1 microgram/ml JH I which also prevents the pupal commitment of the cells. By contrast, USP-2 mRNA increased steadily with the same EC50 as in fourth stage epidermis, irrespective of the presence or absence of JH. Under the same conditions, mRNAs for both EcR-B1 and EcR-A isoforms were up-regulated by 20E, each in its own time-dependent manner, similar to that seen in vivo. These initial mRNA increases were unaffected by the presence of JH I, but those seen after 12 h exposure to 20E were prevented by JH, indicating a difference in response between larvally and pupally committed cells. The presence of JH which maintained larval commitment of the cells also prolonged the half-life of the EcR proteins in these cells. These results indicate that both EcR and USP RNAs are regulated by 20E and can be modulated by JH in a complex manner with only that of USP-2 apparently unaffected.     

crooked legs encodes a family of zinc finger proteins required for leg morphogenesis and ecdysone-regulated gene expression during Drosophila metamorphosis

Drosophila imaginal discs undergo extensive pattern formation during larval development, resulting in each cell acquiring a specific adult fate. The final manifestation of this pattern into adult structures is dependent on pulses of the steroid hormone ecdysone during metamorphosis, which trigger disc eversion, elongation and differentiation. We have defined genetic criteria that allow us to screen for ecdysone-inducible regulatory genes that are required for this transformation from patterned disc to adult structure. We describe here the first genetic locus isolated using these criteria: crooked legs (crol). crol mutants die during pupal development with defects in adult head eversion and leg morphogenesis. The crol gene is induced by ecdysone during the onset of metamorphosis and encodes at least three protein isoforms that contain 12-18 C2H2 zinc fingers. Consistent with this sequence motif, crol mutations have stage-specific effects on ecdysone-regulated gene expression. The EcR ecdysone receptor, and the BR-C, E74 and E75 early regulatory genes, are submaximally induced in crol mutants in response to the prepupal ecdysone pulse. These changes in gene activity are consistent with the crol lethal phenotypes and provide a basis for understanding the molecular mechanisms of crol action. The genetic criteria described here provide a new direction for identifying regulators of adult tissue development during insect metamorphosis.     

Drosophila ecdysone receptor mutations reveal functional differences among receptor isoforms

The steroid hormone ecdysone directs Drosophila metamorphosis via three heterodimeric receptors that differ according to which of three ecdysone receptor isoforms encoded by the EcR gene (EcR-A, EcR-B1, or EcR-B2) is activated by the orphan nuclear receptor USP. We have identified and molecularly mapped two classes of EcR mutations: those specific to EcR-B1 that uncouple metamorphosis, and embryonic-lethal mutations that map to common sequences encoding the DNA- and ligand-binding domains. In the larval salivary gland, loss of EcR-B1 results in loss of activation of ecdysone-induced genes. Comparable transgenic expression of EcR-B1, EcR-B2, and EcR-A in these mutant glands results, respectively, in full, partial, and no repair of that loss.     

The DHR78 nuclear receptor is required for ecdysteroid signaling during the onset of Drosophila metamorphosis

Pulses of ecdysteroids direct Drosophila through its life cycle by activating stage- and tissue-specific genetic regulatory hierarchies. Here we show that an orphan nuclear receptor, DHR78, functions at the top of the ecdysteroid regulatory hierarchies. Null mutations in DHR78 lead to lethality during the third larval instar with defects in ecdysteroid-triggered developmental responses. Consistent with these phenotypes, DHR78 mutants fail to activate the mid-third instar regulatory hierarchy that prepares the animal for metamorphosis. DHR78 protein is bound to many ecdysteroid-regulated puff loci, suggesting that DHR78 directly regulates puff gene expression. In addition, ectopic expression of DHR78 has no effects on development, indicating that its activity is regulated post-translationally. We propose that DHR78 is a ligand-activated receptor that plays a central role in directing the onset of Drosophila metamorphosis.     

Pain. A prelude

The regulatory basis for differences among species in the developmental rate at which successive life stages are reached ("heterochrony") is a subject of much controversy among vertebrate and invertebrate developmental biologists. The heterochrony in development of different insect species is characterized in part by the intercalcation between the embryo and adult of a (varied) number of heteromorphic larval instars. These heteromorphic larval instars exhibit changes of body form from one larval instar to the next, prior to the final metamorphic molt to the pupal form. The intractability of larval heteromorphosis to experimental dissection is due in part to the lack of suitable experimental probes that can test the nature of the coupling of each heterochronically expressed instar-specific program. The epistatic basis of expression of heteromorphic developmental programs was assessed by two-dimensional electrophoretic analysis of hemolymph proteins during the normal and experimentally manipulated feeding stages of the 3rd, 4th, and 5th (final) instar larvae, and during the prepupal stage, of Trichoplusia ni. Of the several hundred protein spots tracked, some were identified that were uniquely detected during a single stage, while others were observed during combinations of certain stages. The nature of coupling of sequential heterochronic expression of these proteins during successive instars or stages was tested by use of a parasite (Chelonus sp.) that injects regulatory material into the host embryo that later causes the subsequent precocious expression of the final instar larval program. Following the expression of a normal 3rd instar pattern, such larvae were observed to omit expression of the 4th instar program, including omission of the proteins heteromorphically specific to that instar, and instead then express an essentially normal final instar pattern. Thus, normal expression of the final instar feeding stage pattern was not invariantly coupled to prior expression of the penultimate instar-specific proteins or pattern. Also, expression of the full program of the final instar feeding stage was epistatic to teh penultimate instar program, i.e., the protein pattern unique to the penultimate larval instar was not co-expressed with the precociously expressed final instar pattern. Larvae developmentally redirected in this manner failed to fully express the final instar prepupal stage pattern of protein expression, due at least in part to failed expression of prepupal ecdysteroids, but this was shown not to arise from omission of any of the first 4 larval instars per se. The nature of the redirections in host development caused by this parasite finally provides means of probing the coupling of successive expression on heteromorphic programs during larval development.     

Steroid regulated programmed cell death during Drosophila metamorphosis

During insect metamorphosis, pulses of the steroid hormone 20-hydroxyecdysone (ecdysone) direct the destruction of obsolete larval tissues and their replacement by tissues and structures that form the adult fly. We show here that larval midgut and salivary gland histolysis are stage-specific steroid-triggered programmed cell death responses. Dying larval midgut and salivary gland cell nuclei become permeable to the vital dye acridine orange and their DNA undergoes fragmentation, indicative of apoptosis. Furthermore, the histolysis of these tissues can be inhibited by ectopic expression of the baculovirus anti-apoptotic protein p35, implicating a role for caspases in the death response. Coordinate stage-specific induction of the Drosophila death genes reaper (rpr) and head involution defective (hid) immediately precedes the destruction of the larval midgut and salivary gland. In addition, the diap2 anti-cell death gene is repressed in larval salivary glands as rpr and hid are induced, suggesting that the death of this tissue is under both positive and negative regulation. Finally, diap2 is repressed by ecdysone in cultured salivary glands under the same conditions that induce rpr expression and trigger programmed cell death. These studies indicate that ecdysone directs the death of larval tissues via the precise stage- and tissue-specific regulation of key death effector genes.     

Effects of the neem tree compounds azadirachtin, salannin, nimbin, and 6-desacetylnimbin on ecdysone 20-monooxygenase activity

The effects of azadirachtin, salannin, nimbin, and 6-desacetylnimbin on ecdysone 20-monooxygenase (E-20-M) activity were examined in three insect species. Homogenates of wandering stage third instar larvae of Drosophila melanogaster, or abdomens from adult female Aedes aegypti, or fat body or midgut from fifth instar larvae of Manduca sexta were incubated with radiolabeled ecdysone and increasing concentrations (from 1 x 10(-8) to 1 x 10(-3) M) of the four compounds isolated from seed kernels of the neem tree, Azadirachta indica. All four neem tree compounds were found to inhibit, in a dose-dependent fashion, the E-20-M activity in three insect species. The concentration of these compounds required to elicit a 50% inhibition of this steroid hydroxylase activity in the three insect species examined ranged from approximately 2 x 10(-5) to 1 x 10(-3).     

Emotions, coping and the need for support in families of children with cancer: a model for psychosocial care

Cloning and characterization of a Choristoneura fumiferana ultraspiracle (Cfusp) cDNA are described. First, a PCR fragment and then a cDNA clone (4.4 kb) were isolated from spruce budworm cDNA libraries. Comparison of the deduced amino acid sequence of this cDNA with the sequences in Genbank showed that this sequence had high homology with the ultraspiracle cDNAs cloned from Drosophila melanogaster (Dmusp), Bombyx mori (Bmusp), Manduca sexta (Msusp), and Aedes aegypti (Aausp). The Cfusp cDNA contained all the regions that are typical for a steroid/thyroid hormone receptor superfamily member. The DNA binding domain or C region was the most conserved sequence among all the usps. The A/B, D, and E regions also showed high amino acid identity with the amino acid sequences of Dmusp, Msusp, Bmusp, and Aausp. The Cfusp 4.5-kb mRNA was present in the embryos, in all larval stages, and in the pupae. The Cfusp mRNA levels in the midgut increased during the sixth-instar larval development and reached peak levels during the ecdysteroid raises for the pupal molt. However, Cfusp mRNA levels remained unchanged in the midgut of fifth-instar larvae, and in the epidermis and fat body of sixth-instar larvae indicating both a tissue- and stage-specific regulation of Cfusp mRNA expression.     

Survival, developmental and morphogenic deficiencies of Parasarcophaga argyrostoma (Diptera: Sarcophagidae) induced by the biosynthesis inhibitor, chlorfluazuron (IKI-7899)

To evaluate the activity of benzoylphenyl urea chitin biosynthesis inhibitor chlorfluazuron (IKI-7899) against Parasarcophaga argyrostoma, seven doses were topically applied (once) onto early third (last) instar larvae, puparia, or newly formed pupa: 150, 100, 50, 10, 1.0, 0.5, and 0.25 microgram/insect. After topical treatment of last instar larvae, the highest mortality was caused by both higher doses and the lowest mortality was caused by the lowest dose. The lethal activity of IKI-7899 as pronouncedly decreased as the treatment was lately carried out (at the puparial time). IKI-7899 failed to cause cumulative mortality because no pupal or adult mortalities were observed, irrespective of the time of treatment. Treated larvae suffered the action of IKI-7899 because they had decreased weight gain. Except the lowest dose, the weight gain of larvae inversely correlated with the dose-levels. IKI-7899 prolonged not only the larval duration but also the pupal duration after topical treatment of last instar larvae with doses 50-0.25 micrograms/larva. With no exception, all doses topically applied onto puparia or newly formed pupae enhanced pupae to live longer. Topical application onto last instar larvae resulted in different degrees of reduction of pupation rate, but IKI-7899 could not affect the pupal morphogenesis after larval treatment except by its highest dose which led to 8.33% pupal deformities and 7.69% larval-pupal intermediates. The dose 100 micrograms/larva topically applied onto last instar larvae detained 7.69% of what known as "permanent larvae" which suffered the action of the compound along 16 days and eventually perished without any external feature of puparium formation. A metamorphic effect of IKI-7899 pronouncedly appeared in the adult stage. Three higher doses completely arrested the adult flies. Topical application of the compound onto prepupae did not greatly reduce the pupation rate especially at the doses 50, 10 and 1.0 micrograms/puparium. The dose 50 micrograms/puparium was only the dose halting the pupal moulting program because 7.14% of permanent prepupae remained about 12 days and then died. In respect to adult emergence, the highest dose led to zero rate and the lowest dose allowed to all pupae to emerge without malformation.     

The major excretory/secretory protease from Lucilia cuprina larvae is also a gut digestive protease

The larvae of the fly Lucilia cuprina excrete or secrete a chymotrypsia (LCTb) onto the skin of sheep to facilitate the establishment of the larval infestation. A combination of immunoblotting and RT-PCR approaches has established that this protease is also a gut digestive protease. LCTb is synthesized primarily in the cardia, a small highly specialized organ located at the anterior end of the midgut and by midgut cells. There is also some expression by the hindgut but no expression by salivary glands. Excretion of LCTb with waste products or regurgitation of the gut contents of the larvae may explain how this protease is transferred from the larval gut onto ovine skin. LCTb is first expressed in eggs and constitutively expressed throughout each larval instar, but is not expressed in pupae or adult flies. It is concluded that LCTb could be involved in the establishment of larvae on sheep skin as well as acting as a general gut digestive enzyme.     

Central projections of persistent larval sensory neurons prefigure adult sensory pathways in the CNS of Drosophila

We have used a GAL4 enhancer-trap line driving the expression of a lacZ construct to examine the reorganisation of an identified group of proprioceptive sensory neurons during metamorphosis in Drosophila. The results show that whilst most larval sensory neurons degenerate during the first 24 hours of metamorphosis a segmentally repeated array of 6 neurons per segment persists into the adult stages to become functional adult neurons. These sensory neurons retain their axonal projections in the central nervous system intact and unchanged throughout. The adult sensory neuron axons enter the central nervous system at around 44 hours after puparium formation. Most of these axons grow along the pathways defined by the persistent larval sensory axons. The ordering of the adult sensory projections is, therefore, established upon the larval pattern of projections. The possibility that the larval neurons act as guidance cues for organising the ordered arrays of sensory neurons is discussed.     

Mode of action of lufenuron on larval cat fleas (Siphonaptera: Pulicidae)

Adult cat fleas, Ctenocephalides felis (Bouché), were fed suboptimal in vitro concentrations of lufenuron in blood to allow hatching of flea larvae for cytological study. At concentrations of 0.125, 0.25, and 0.5 ppm, larval hatch was 64, 15, and 4%, respectively. Larvae hatching from eggs laid by adults fed lufenuron at concentrations of 0.025, 0.08, or 0.125 ppm did not differ significantly from the control. However, many larvae from the 0.08-ppm group and higher concentrations died during the 1st instar. Examination of these larvae revealed that they were dying from desiccation caused by bleeding from microscopic lesions in the cuticle or the inability to complete the molt to the next instar. Electron micrographs showed that lufenuron often disrupted formation of the endocuticle resulting in the deposition of an amorphous mass of randomly oriented chitin microfibrils. Other larvae formed normal endocuticle but were unable to digest the old endocuticle or produce new procuticle after apolysis. Failure of larvae to digest old cuticle or form new cuticle was caused by degeneration of the epidermal cells needed for the synthesis of molting fluid and chitin.