Juvenile hormone: effects on a higher dipteran.

Injection of dl-juvenile hormone or C(17) methyl ester into Sarcophaga bullata larvae prevents puparium formation or arrests development at about the 3rd day of pupal-adult development. Topical application to the abdomens of young pupae results in the secretion of a second pupal cuticle. This is the first reported morphogenetic effect of juvenile hormone on a fly.

Insect prothoracicotropic hormone: evidence for two molecular forms.

In an insect, the tobacco hornworm Manduca sexta, the cerebral neuropeptide prothoracicotropic hormone (PTTH), the primary effector of postembryonic development, exists as two molecular forms. These two PTTH's elicit characteristic in vitro dose responses of activation of prothoracic glands from different developmental stages, an indication that during development the glands change in their sensitivity to the neurohormones. Both PTTH's are active in a specific in situ bioassay. Since they may be released in situ at stage-specific times to evoke distinctly different developmental responses, the PTTH neuroendocrine axis appears to be an effective system for determining the functions of molecular forms of a neurohormone in the regulation of growth and development.

Evidence for the existence of two forms of RNA polymerases I and II in insect wing epidermis.

DNA-dependent RNA polymerases were solubilized from developing wings of the oak silkmoth, Antheraea pernyi, and partially purified by ion-exchange chromatography and sucrose gradient sedimentation. Four enzyme species were resolved on the basis of chromatographic behavior, divalent cation requirements, ionic strength optima, template preference and alpha-amanitin sensitivity. Each class (i.e. RNA polymerase I and II) was present in two forms termed IA, IB and IIA, IIB on the basis of their elution pattern from the column. Both class I enzymes were sensitive to high concentrations of alpha-amanitin but this may be due to general toxicity rather than specific inhibition. The intraclass variants did not differ significantly in enzymatic properties although form IIB was more sensitive to alpha-amanitin (50% inhibition at 2 . 10(-9) M) than form IIA (3 . 10(-8)M).

Developmental arrest and ecdysteroid deficiency resulting from mutations at the dre4 locus of Drosophila.

Loss-of-function mutations of the dre4 gene of Drosophila melanogaster caused stage-specific developmental arrest, the stages of arrest coinciding with periods of ecdysteroid (molting hormone) regulated development. Nonconditional mutations resulted in the arrest of larval development in the first instar; embryogenesis was not impaired, and mutant larvae were behaviorally normal and long-lived. At 31 degrees the temperature-sensitive dre4e55 allele caused the arrest of larval development in the first or second instars. When upshifted to 31 degrees at various times during development, dre4e55 mutants exhibited nonpupariation of third-instar larvae, failure of pupal head eversion, failure of adult differentiation, or noneclosion of pharate adults. Under some temperature regimens second-instar larvae pupariated precociously without entering the normally intervening third-instar. Nonpupariation and defects in metamorphosis were associated with the reduction or elimination of ecdysteroid peaks normally associated with late-larval, prepupal, pupal and pharate adult development. Ecdysteroid production by larval ring glands from dre4e55 hemizygous larvae was suppressed after 2 hr of incubation in vitro at 31 degrees, indicating autonomous expression of the dre4 gene in the ring gland. We postulate that the dre4 gene is required for ecdysteroid production at multiple stages of Drosophila development and that the pathologies observed in dre4 mutants reflect developmental consequences of ecdysteroid deficiency.

The genetics of the Dras3-Roughened-ecdysoneless chromosomal region (62B3-4 to 62D3-4) in Drosophila melanogaster: analysis of recessive lethal mutations.

The genetic organization of interval 62B3-4 to 62D3-4 on the Drosophila third chromosome was investigated. The region (designated DRE) includes four known loci: Roughened (R; 3-1.4), defined by a dominant mutation disrupting eye morphology; the nonvital locus Aprt, structural gene for adenine phosphoribosyltransferase; Dras3, a homolog of the vertebrate ras oncogene; and 1(3)ecdysoneless (1(3)ecd), a gene that has been implicated in the regulation of larval molting hormone (ecdysteroid) synthesis. Overlapping chromosomal deletions of the region were generated by gamma-ray-induced reversion of the R mutation. Recessive lethal mutations were isolated based upon failure to complement the recessive lethality of Df(3L)RR2, a deletion of the DRE region that removes 16-18 polytene chromosome bands. A total of 117 mutations were isolated following ethyl methanesulfonate and gamma-ray mutagenesis. These and two additional define 13 lethal complementation groups. Mutations at two loci were recovered at disproportionately high rates. One of these loci is preferentially sensitive to radiation-induced mutational alterations. Additionally, an unusually low recovery rate for cytologically detectable rearrangement breakpoints within the gamma-ray-sensitive locus suggests that an interval of the DRE region closely linked to the R locus may be dominantly sensitive to position effects. Lethal phase analysis of mutant hemizygotes indicates that a high proportion of DRE-region loci (11 of 13) are necessary for larval development. Mutations in five loci cause predominantly first-instar larval lethality, while mutations in four other loci cause predominantly second-instar lethality. Mutations in two loci cause late-larval lethality associated with abnormal imaginal disc development. A temperature-sensitive allele of one newly identified complementation group blocks ecdysteroid-induced pupariation. This developmental block is overcome by dietary 20-hydroxyecdysone, suggesting that a second locus in the region in addition to l(3)ecd may play a role in the regulation of late larval ecdysteroid levels.

Prothoracicotropic hormone stimulated extracellular signal-regulated kinase (ERK) activity: the changing roles of Ca(2+)- and cAMP-dependent mechanisms in the insect prothoracic glands during metamorphosis.

The synthesis of ecdysteroids by the lepidopteran prothoracic gland is regulated by a brain neuropeptide hormone, prothoracicotropic hormone (PTTH). In Manduca sexta glands, PTTH stimulates several events including Ca(2+) influx, Ca(2+)-dependent cAMP generation and the activation of several protein kinases. In the present study, the path by which PTTH stimulates extracellular signal-activated regulated kinase (ERK) phosphorylation was investigated using PTTH and second messenger analogs. The results indicate that Ca(2+)-dependent processes, other than cAMP generation, play the major role in PTTH stimulation of ERK phosphorylation in larval prothoracic glands, that cAMP-dependent events increase in importance during later development and that PTTH-stimulated ERK phosphorylation is highest in larval glands. The decline in PTTH-stimulated ERK phosphorylation associated with metamorphosis results from decreased ERK levels and an increased basal rate of ERK phosphorylation. The data suggest that the role or importance of components of the PTTH signal transduction cascade are not fixed and can change during development.

Prothoracicotropic hormone-regulated expression of a hsp 70 cognate protein in the insect prothoracic gland.

In Manduca sexta, ecdysteroids coordinate molting and metamorphosis of insects and are produced by the prothoracic glands under the acute control of the brain neuropeptide prothoracicotropic hormone (PTTH). PTTH stimulates rapid ecdysteroidogenesis accompanied by specific increases in the synthesis and accumulation of three proteins, including one with M(r) = 70 kDa. This 70-kDa protein is a constitutively expressed member of the heat shock protein 70 family (hsc 70). Levels of this hsc 70 vary in a prothoracic gland-specific manner during development as does its PTTH-stimulated synthesis when assayed in vitro. The accumulation of hsc 70 may be regulated by abrupt changes in its turnover rate. The PTTH-stimulated increase in hsc 70 synthesis is dependent upon both translational and transcriptional events. Hsc 70 expression in the prothoracic gland may be required for changes in gland growth, e.g., protein content, that underlie alterations in ecdysteroid production.

Cloning of a beta1 tubulin cDNA from an insect endocrine gland: developmental and hormone-induced changes in mRNA expression.

A rapid increase in ecdysteroid hormone synthesis results when the insect prothoracic gland is stimulated with prothoracicotropic hormone (PTTH), a brain neuropeptide hormone. PTTH also stimulates the specific synthesis of several proteins, one of which is a beta tubulin. To further understand the possible roles of beta tubulin in the prothoracic gland, beta tubulin cDNA clones were isolated from a tobacco hornworm (Manduca sexta) gland cDNA library. Sequence analysis indicated that these clones were assignable to the beta1 tubulin isoform. Gland beta1 tubulin mRNA levels during the last larval instar and early pupal-adult development exhibited peaks that coincided with peaks in ecdysteroid synthesis. Manipulations of the glands hormonal milieu showed that beta1 tubulin mRNA levels respond to 20 hydroxyecdysone and PTTH. The data also support our earlier proposal that the prothoracic gland beta1 tubulin gene is ubiquitously expressed but exhibits tissue- and developmental-specific regulation of transcription and translation.

Occurrence, quaternary structure and function of G protein subunits in an insect endocrine gland.

The occurrence, structure and function of the alpha and beta subunits of GTP-binding proteins (G proteins) were investigated in the Manduca sexta prothoracic gland, a tissue which possesses a hormonally regulated adenylate cyclase. Subunit-specific antibodies were utilized in immunoblotting studies of tissue from Manduca prothoracic glands, brain, eyes and antennae, and compared to the substrates present in the heads of Drosophila, as well as in a mammalian cell line. All Manduca tissues examined showed putative G beta subunits of 37 and 38 kDa, an unidentified alpha subunit of 41 kDa, in addition to an eye specific alpha subunit of 42 kDa. Manduca tissues also produced putative Gs alpha subunits of 48 and 51 kDa which were coupled to prothoracic gland adenylate cyclase as demonstrated by immunoprecipitation. Prothoracic gland G proteins have a definite and limited quaternary structure, consistent with a heterotrimeric model, as demonstrated by crosslinking of prothoracic gland membrane preparations followed by immunoblotting. These studies also yielded data on relative titers of alpha subunits, and suggest that Gs alpha is present in lower amounts than other alpha subunits. The G protein subunits studied in the prothoracic gland appear strikingly similar in molecular weight, function and structure to their mammalian counterparts.

Polyamines modulate multiple protein phosphorylation pathways in the insect prothoracic gland.

Multiple endogenous substrates phosphorylated by four distinct protein kinases were identified in particulate and cytosolic fractions from the larval prothoracic gland of the tobacco hornworm, Manduca sexta. Three prominent particulate-associated phosphoprotein substrates (19, 21, and 34 kDa) were of particular interest. The in vitro phosphorylation of the 19 and 21 kDa peptides was markedly enhanced by cAMP, Ca2+/calmodulin, as well as Ca2+/phospholipids, presumably via cAMP-dependent protein kinase (cAMP-PK), Ca2+/calmodulin-dependent protein kinase (Ca2+/CaM-PK), and protein kinase C (PKC), respectively. The polyamine spermine markedly inhibits both PKC- and cAMP-PK-mediated phosphorylation of the 19 and 21 kDa peptides but had no effect on the Ca2+/CaMP-PK-mediated phosphorylation. Spermine also inhibits the phosphorylation of the 34 kDa peptide via cAMP-PK but does not affect PKC-promoted phosphorylation. In contrast to this differential inhibition of phosphorylation by a polyamine, four cytosolic and three particulate-associated peptides from the prothoracic glands undergo enhanced phosphorylation in the presence of spermine, presumably by stimulating casein kinase II activity. Therefore, polyamines appear to have multiple effects on protein phosphorylation pathways in this important endocrine gland, perhaps representing an important new regulatory control mechanism.

mRNA populations during wing development in the silkmoth Antheraea polyphemus.

Pupal wing tissue of the American silkmoth Antheraea polyphemus has been used as a model system to study 20-hydroxyecdysone and juvenile hormone control of cuticle protein synthesis. Juvenile hormone does not affect either the content or rate of synthesis of RNA and protein of the wing tissue. both of which show linear increases during the first few days of hormonal treatment. Based on the fractionation of total RNA on oligo-dT columns the percent of mRNA remains the same throughout development after both hormone treatments. However, both the amount of poly-A+ RNA in the wing tissue, and its content of poly-A show considerable increases as a function of development. The products of translation of the various poly-A+ RNA populations in the cell-free wheatgerm system have been analyzed by one- and two-dimensional gel electrophoresis and fluorography. Qualitative changes occur during the first 24 h; the production of a mRNA coding for a protein of approx. 40 000 dalton is stimulated and the production of a mRNA coding for a protein of 29 000 dalton is greatly reduced. Only a few differences are observed between samples from the 2 hormone treatments. Over the next 5-15 days of development mainly quantitative changes are observed. Juvenile hormone application results in quantitative changes in specific mRNAs, but no new mRNAs unique to juvenile hormone action are observed. The data are consistent with the concept that in altering the epidermal developmental program, juvenile hormone is apparently modulating the action of 20-hydroxyecdysone.

Spermine and polylysine enhanced phosphorylation of calmodulin and tubulin in an insect endocrine gland.

Spermine-stimulated and heparin-inhibited phosphorylation of both exogenous casein and endogenous protein substrates of the prothoracic gland were measured in prothoracic gland cytosolic fractions from fifth instar larvae and early pupae of the tobacco hornworm, Manduca sexta. The results reveal a striking increase in casein kinase II (CKII) activity, i.e. approximately 3-fold above basal level in the presence of 5 mM spermine, with the highest activity exhibited by gland fractions from day 0-2 larvae, newly pupated animals and day 1 pupae. These results were verified by the results from Western blot analysis using a CKII alpha-subunit specific antibody and a 10 a.a. synthetic peptide that is a specific substrate for CKII. Several endogenous proteins were found to be substrates for CKII when assayed in the presence of spermine or polylysine. A 19 kDa peptide was shown to be calmodulin (CaM) by using the purified Manduca brain CaM as an indicator, and was only phosphorylated in the presence of polylysine. A 52 kDa protein was identified as tubulin by immunoprecipitation with a tubulin-specific monoclonal antibody, and was shown to be phosphorylated in the presence of spermine and polylysine. The possible roles of phosphocalmodulin and phosphotubulin are discussed in the context of prothoracic gland function.

Activation of an extracellular signal-regulated kinase (ERK) by the insect prothoracicotropic hormone.

Ecdysteroid hormones are crucial in controlling the growth, molting and metamorphosis of insects. The predominant source of ecdysteroids in pre-adult insects is the prothoracic gland, which is under the acute control of the neuropeptide hormone prothoracicotropic hormone (PTTH). Previous studies using the tobacco hornworm, Manduca sexta, have shown that PTTH stimulates ecdysteroid synthesis via a series of events, including the activation of protein kinase A and the 70 kDa S6 kinase (p70(S6k)). In this study, PTTH was shown to stimulate also mitogen-activated protein kinase (MAPK) phosphorylation and activity in the Manduca prothoracic gland. The MAPK involved appears to be an extracellular signal-regulated kinase (ERK) homologue. The ERK phosphorylation inhibitors PD 98059 and UO 126 blocked basal and PTTH-stimulated ERK phosphorylation and ecdysteroid synthesis. PTTH-stimulated ERK activity may be important for both rapid regulation of ecdysteroid synthesis and for longer-term changes in the size and function of prothoracic gland cells.

DNA-dependent RNA polymerase activity in silkmoth-wing epidermis after hormone treatment.

DNA-dependent RNA polymerase activity of wing epidermal tissue from the silkmoth, Antheraea polyphemus, has been studied after treatment of pupae with either molting hormone 20-hydroxyecdysone or 20-hydroxyecdysone and juvenile hormone. Enzyme activity has been measured both on endogenous template in isolated nuclei and on exogenous template after solubilization and correlated with transcriptional activity measured as the incorporation of [3H]uridine into RNA. Within 4 h of either hormonal regimen, increases in nuclear transcriptional activity for enzymes I and II are observed. Maximal nuclear activity for both enzyme classes was observed at 26 h. Solubilized enzyme activity, on the other hand, increased continuously up to 144 h. The increase in enzyme activity at 26 h, and probably earlier, is dependent on both RNA and protein synthesis, indicating that the increase is not a consequence of the activation of inactive molecules, but requires the synthesis of either new enzyme molecules or effector molecules. Application of 20-hydroxyecdysone + juvenile hormone does not significantly affect nuclear RNA polymerase activity, rates of RNA synthesis or even RNA content during the first 26 h. However, JH causes significant diminution in the rise of solubilized activity observed with 20-hydroxyecdysone. This reduction is not a consequence of diminished protein content. Therefore, the number of active RNA polymerase molecules appears not to directly correspond to the rate of RNA synthesis.

Involvement of cyclic AMP-dependent protein kinase in prothoracicotropic hormone-stimulated ecdysone synthesis.

Prothoracicotropic hormone (PTTH) is a brain neuropeptide that stimulates the prothoracic glands to synthesize ecdysone, an event that leads to insect molting. Both cyclic AMP (cAMP) and calcium have been implicated in PTTH action, with current evidence favoring cAMP as the messenger directly regulating ecdysone synthesis. To further define the role of cAMP in PTTH action, the activity of cAMP-dependent protein kinase (cAMP-PK) was examined in prothoracic glands from two developmental stages of the tobacco hornworm, Manduca sexta (day 3 fifth instar larvae and day 0 pupae). Prothoracic glands at each of these stages of development possess two forms of cAMP-PK which resemble the vertebrate type I and type II isozymes, with the latter being the predominant form (greater than 90%). Marked developmental differences exist in the degree of activation of soluble cAMP-PK following in vitro exposure of the prothoracic glands to PTTH. In larval glands, soluble cAMP-PK is activated within 3-10 min of initial exposure to doses of PTTH that stimulate ecdysone synthesis. By contrast, activation of soluble cAMP-PK in pupal glands occurs only when PTTH is administered in the presence of a phosphodiesterase inhibitor. Developmental differences in the activation of cAMP-PK by PTTH were qualitatively identical to previously observed differences in PTTH-stimulated accumulation of intracellular cAMP. The results suggest an involvement of soluble cAMP-PK in the response of day 3 fifth instar larval prothoracic glands to PTTH, but indicate a difference in the nature, intracellular location, or time course of activation, of hormone-sensitive protein kinase in day 0 pupal glands.

Calcium-cyclic AMP interactions in prothoracicotropic hormone stimulation of ecdysone synthesis.

The calcium-dependence of ecdysone synthesis by the insect prothoracic glands was examined in vitro using glands from day 0 pupae of the tobacco hornworm, Manduca sexta. Stimulation of ecdysone synthesis by the cerebral neuropeptide, prothoracicotropic hormone (PTTH), requires extracellular calcium; peptide-stimulated steroidogenesis is blocked by omission of calcium or by addition of the calcium antagonist lanthanum. By contrast, basal synthesis of ecdysone is not calcium-dependent. A stimulatory, as opposed to simply a permissive, role for calcium is indicated by the ability of the calcium ionophore A23187 to mimic the steroidogenic effects of PTTH. Agents that act by increasing the intracellular levels of cAMP (dibutyryl cAMP, 1-methyl-3-isobutylxanthine, forskolin) enhance ecdysone synthesis equally well in the presence or absence of extracellular calcium, indicating that the site of action of the cation in the steroidogenic pathway occurs prior to that of cAMP. Both PTTH and A23187 enhance the formation of cAMP, as measured by the conversion of [3H]ATP to [3H]cAMP, in a manner absolutely dependent upon the presence of extracellular calcium. The results suggest sequential roles for calcium and cAMP in PTTH-stimulated steroidogenesis by the insect prothoracic glands. A model is presented in which the peptide stimulates cAMP formation in a calcium-dependent manner, with the cyclic nucleotide in turn enhancing ecdysone synthesis.

The influence of hemolymph-binding protein on juvenile hormone stability and distribution in Manduca sexta fat body and imaginal discs in vitro.

The wing discs and fat body of Manduca sexta larvae contain enzymes (i.e. carboxylesterase and epoxide hydratase) that can convert the C18 juvenile hormone (JH) to the acid, diol and acid diol. No evidence of oxidative degradation was noted. In vitro studies suggest that JH can be compartmentalized within the cells of the fat body where it is less accessible to degradative mechanisms. Experiments utilizing a hemolymph-binding protein fraction (BPF) in vitro with fat body and imaginal discs indicate that the BPF retards the uptake of JH by tissues and its subsequent degradation by tissue enzymes. BPF also appears to protect JH from degradation by enzymes released into the medium. By these mechanisms the insect can maintain elevated JH titers for relatively long periods. Binding protein may also keep JH in solution in the hemolymph allowing its rapid distribution throughout the insect. The data suggest that the binding protein plays a key role in maintaining juvenile hormone titers.

Ecdysone 20-monooxygenase activity during larval-pupal development of Manduca sexta.

Profiles of ecdysone 20-monooxygenase (E-20-M) activity in the fat body and midgut of Manduca sexta were determined during larval-pupal development. The E-20-M activities for both tissues were found to exhibit a single major fluctuation during this 10-day period of development: fat body, a 10-fold fluctuation with peak activity on day 4; midgut, a 60-fold fluctuation with peak activity on day 5. Substrate kinetics revealed that the apparent Km values of fat body and midgut monooxygenases for ecdysone were fairly constant during the instar, 2.42 X 10(-7) M and 4.67 X 10(-7) M, respectively. By contrast, the monooxygenase Vmax values in each tissue fluctuated in a manner both quantitatively and temporally coincident with the fluctuations in enzyme activity. These findings suggest that changes in E-20-M activity are a function of changes in the titer of the enzyme. The possible developmental significance of the fluctuations in E-20-M activity are discussed.

Insect prothoracic glands: a role for cyclic AMP in the stimulation of alpha-ecdysone secretion.

The prothoracic glands of the tobacco hornworm, Manduca sexta, were studied to determine if cyclic AMP is involved in the regulation of alpha-ecdysone secretion. Culturing glands in the presence of the phosphodiesterase inhibitors, aminophylline and 1-methyl-3-isobutylxanthine, caused a greater than 2-fold stimulation of ecdysone secretion while cyclic AMP alone was ineffective. Based on a dose-response analysis, 1-methyl-3-isobutylxanthine was 200 times more potent than aminophylline. Measurements of endogenous prothoracic gland cyclic AMP during the fifth larval instar demonstrated that dramatically increased levels preceded the increase in in vitro ecdysone-secretory ability. The data suggest that cyclic AMP may act as a second messenger in the stimulation of prothoracic gland alpha-ecdysone secretion by the prothoracicotropic brain hormone.

Woc (without children) gene control of ecdysone biosynthesis in Drosophila melanogaster.

The first step in ecdysteroidogenesis, i.e. the 7,8-dehydrogenation of dietary cholesterol (C) to 7-dehydrocholesterol (7dC), is blocked in Drosophila melanogaster homozygous woc (without children) third instar larval ring glands (source of ecdysone). Unlike ring glands from wild-type D. melanogaster larvae, glands from woc mutants cannot convert radiolabelled C or 25-hydroxycholesterol (25C) to 7dC or 7-dehydro-25-hydroxycholesterol (7d25C) in vitro, nor to ecdysone (E). Yet, when these same glands are incubated with synthetic tracer 7d25C, the rate of metabolism of this polar Delta(5,7)-sterol into E is identical to that observed with glands from comparably staged wild-type larvae. The absence of this enzymatic activity in vivo is probably the direct cause of the observed low whole-body ecdysteroid titers in late third instar homozygous mutant larvae, the low ecdysteroid secretory activity in vitro of brain-ring gland complexes from these animals, and the failure of the larvae to pupariate (undergo metamorphosis). Oral administration of 7dC, but not C, results in a dramatic increase in ecdysteroid production both in vivo and in vitro by the woc mutant brain-ring gland complexes and affects a partial rescue to the beginning of pupal-adult development, but no further, despite elevated whole-body ecdysteroid titers. Data previously reported (Wismar et al., 2000) indicate that the woc gene encodes a zinc-finger protein that apparently modulates the activity of the 7,8-dehydrogenase.