Pharate Adult Stage Research Articles

INVITATION PAPER (C.P. ALEXANDER FUND): ENDOCRINE REGULATION OF MALE ACCESSORY GLAND DEVELOPMENT AND ACTIVITY

AbstractFor most species, the male accessory glands are mesodermal derivatives. Their form is as varied as are the functions of the secretion they produce. The post-embryonic development and differentiation of the glands, like those of other tissues, appear to be regulated by the interaction of juvenile hormone (JH) and ecdysteroid, the former inhibiting and the latter promoting these processes. Post-eclosion accessory gland activity (i.e. production of secretion), for most species examined, is regulated by JH. However, the precise mechanism of control and the site of action are not at all clear. A limited amount of in vitro work indicates adirectaction of JH on the accessory glands though only forDrosophila melanogasterMeigen has a JH-receptor protein been identified. The JH promotes the synthesis ofspecificproteins in the accessory gland secretion, and two proposals have been made for the site and mode of action of the hormone. In the first it is suggested that the hormone affects membrane permeability, thereby influencing the supply of protein precursors; in the second a more direct action of JH at the gene level is proposed, namely, the promotion of translation.The involvement of ecdysteroids in accessory gland activity is virtually unexplored though there have been a few demonstrations that these hormones can stimulate protein synthesis. An hypothesis worth further consideration suggests that ecdysteroids may have a role in those species that eclose in a sexually mature condition, i.e. where the accessory glands have both differentiated and produced their secretion in the pupal or pharate adult stage, presumably in the absence of JH.

A monoclonal antibody to a 56 kDa neurosecretory polypeptide from the posterior lateral neurosecretory cells of the housefly, Musca domestica

A protocol was developed for generating monoclonal antibodies (MABs) to neurosecretory cells (NSCs) of the housefly, Musca domestica, using head extract as immunogen. A panel of MABs to housefly NSCs was produced using a 3-tier screening involving enzyme-linked immunosorbent assay (ELISA), western blot, and immunocytochemistry. One clone designated 01C10 was shown to immunostain the posterior lateral NSCs of the adult housefly brain. Immunostaining of neurohemal organs, corpora cardiaca (CC), was restricted to the late pharate adult stage and the first 6 h of adult eclosion. Loss of immunostained material from CC indicated its release into the hemolymph. Western blot studies revealed that 01C10 MAB immunoreacted with a 56 kDa polypeptide in the brain, corpora cardiaca/ corpora allata (CC/CA) complex of newly emerged adults and hemolymph collected 1 h after adult eclosion. These results strongly suggested the neurohormonal nature of the 01C10-immunoreactive polypeptide. We speculated the 56 kDa polypeptide to be bursicon based on a HPLC separation of hemolymph proteins from newly emerged adults which showed the co-elution of bursicon activity and the 56 kDa polypeptide in the same fractions.

A comparison of cuticle deposition during the pre- and posteclosion stages of the adult weevil, Anthonomus grandis Boheman (Coleoptera : Curculionidae)

An ultrastructural analysis of cuticle deposition before and after adult eclosion of the cotton boll weevil, Anthonomus grandis Boheman (Coleoptera : Curculionidae), is made to monitor the intra- and extracellular events that accompany the shift in cuticle architecture. A typical 5-layered epicuticle and a multi-lamellate procuticle are deposited during the pharate adult stage. Secretion of the epicuticle and the procuticle begins about 2 and 4 days after pupation, respectively. Following eclosion, a lattice-like endocuticle is secreted in the form of layers of parallel rod- or beam-shaped macrofibers. Deposition of endocuticle over the first week after emergence, is at a rate of 3 or 4 layers per day. The imaginal endocuticle accounts for the major portion of the cuticle mass as there is about a 14-fold increase in sclerite thickness and an overall 4-fold increase in non KOH-extractable extskeletal mass during the first week after emergence. Extensive cytoskeletal and surface remodeling plus a change in secretory product packaging occurs at the apical region of the epidermal cells upon shifting to deposition of the endocuticle. Intralayer orientation of the macrofibers is under cellular control and is accomplished by the formation of templates consisting of membrane placque-bearing, canal-like depressions on the apical surface extending across cell borders. Comparisons of cuticle sections to simulated plots drawn via computer graphics, show that each successive layer of macrofibers is rotated with respect to the overlying layer by an angle of about 72°. Except for vertical columns of cuticular fibers that support pore canals, microfibril orientation within the procuticle/exocuticle generally follows the Bouligand model for a typical lamellate arthropod cuticle. Direct cellular control over the interlaminar orientation of the microfibrils forming the procuticle could not be discerned in this study.

Mutations in the proximal region of the optomotor-blind locus of Drosophila melanogaster reveal a gradient of neuroanatomical and behavioral phenotypes.

Mutations in the complex optomotor-blind (omb) gene locus (4C4-6) lead to a number of different phenotypes in various tissues of the adult Drosophila melanogaster fly. At the core of the locus lies a lethal complementation group, named l(1)omb, whose mutations cause larval and pupal lethality. Some 40% of all males hemizygous for lethal omb alleles develop to the pharate adult stage. These flies can be rescued from the pupal case and show a severe disturbance in optic lobe development. The recessive viable allele In(1)ombH31 reduces the optomotor response in walking flies and during stationary flight of tethered flies. At the neuroanatomical level, these animals lack a subset of lobula plate giant neurons (LPGNs), which are thought to mediate optomotor behavior. Chromosomal aberrations deleting the proximal, non transcribed part of the locus complement the lethality, but still cause neuroanatomical and optomotor defects. Analysis of different allelic combinations of such mutations, in which increasing amounts of DNA downstream of the transcribed region are removed, reveals a step gradient of increasing severity of the neuroanatomical defects and behavioral phenotypes. On this basis the 3'-regulatory region is divided into three domains each having specific effects on optic lobe development.

Developmental profiles of epidermal mRNAs during the pupal-adult molt of Tenebrio molitor and isolation of a cDNA clone encoding an adult cuticular protein: Effects of a juvenile hormone analogue

Changes in translatable mRNAs from the wing epidermis of the Coleoptera Tenebrio molitor have been investigated during metamorphosis by analysis of in vitro translated products. Striking differences between the patterns obtained from mRNAs extracted during pupal and adult cuticle secretion indicated that a drastic change in gene expression occurs during the pupal-adult transition. In addition to these stage-specific modifications, the mRNA patterns changed within each cuticular synthesis program (pupal or adult), especially at ecdysis. After tritiated leucine incorporation, some of the major radiolabeled cuticular proteins showed similar changes suggesting that the sequential appearance of mRNAs corresponds to sequential deposition of cuticular proteins. In supernumerary pupae obtained after juvenile hormone analogue (JHA) application on newly ecdysed pupae, translatable mRNA were very similar to those of pharate pupae. The JHA seemed, therefore, to prevent the expression of the adult program. By immunoblotting in vitro translated products with a monoclonal antibody recognizing an adult-specific cuticular protein, the developmental profile of the corresponding mRNA was studied. This mRNA was detected in anterior wing epidermis during the first 80 hr of the pharate adult stage. Using the same antibody, a cDNA clone was isolated from epidermal mRNA. The hybrid selected mRNA coded for only one protein with an apparent MW of 22 kDa which was, furthermore, recognized by the antibody. The Northern blot analysis performed with the clone confirmed the Western blot analysis of the in vitro translation products. JHA application at the beginning of the pupal-adult reprograming prevented the appearance of this mRNA; however, this transcript was present during the following molting cycle. This reversibility of the JHA action was confirmed by immunogold labeling of the cuticles formed in treated animals.

Developmental and metabolic changes induced by anoxia in diapausing and non-diapausing flesh fly pupae

Anaerobic metabolism was compared in nondiapausing (ND) and diapausing (D) pupae of the flesh fly,Sarcophage crassipalpis using in vivo13C NMR spectroscopy. Anoxia-induced changes in the development of ND and D pupae were correlated with oxidative metabolism and mitochondrial integrity. ND pupae tolerated 1 day of anoxia without any obvious developmental effect, while D pupae tolerated up to 6 days of anoxia. Longer exposure to anoxia (3 days in ND pupae and up to 14 days in D pupae) allowed development to the pharate adult stage but precluded eclosion. Four-day anoxia applied to ND pupae during 4–6 days post-pupariation arrested development in a stage indistinguishable from diapause. This morphological stasis was accompanied by 80% suppression of oxidative metabolism and a 100% increase in glycerol concentration. However, unlike a true diapause, this arrest could not be terminated with 20-hydroxyecdysone or hexane. Four-day anoxia treatment applied to D pupae stimulated development and raised their oxygen consumption. The anoxia-induced changes in oxidative metabolism were not accompanied by mitochondrial changes. Exposure to 95%PO2 atmosphere had no apparent developmental or metabolic effects on ND or D pupae. Major metabolites (lipids, trehalose, glycogen, glycerol, glutamine, and alanine) were detected in the ND and D pupae but their rates of turnover differed. Anoxia induced synthesis of glycerol and alanine in both D and ND pupae. Injected labeled glucose was incorporated primarily into trehalose and glycogen by both D and ND pupae. The rate of incorporation in ND pupae was approximately twice that observed in D pupae. Anoxia resulted in glycerol and alanine synthesis in both groups of pupae, but more glycerol was labeled in ND pupae and more alanine in D pupae. Glycogen and trehalose were depleted in the D pupae under anoxia. Cold acclimation had no effect on the steady-state or rate of synthesis of metabolites.

Cold shock and heat shock: a comparison of the protection generated by brief pretreatment at less severe temperatures

Abstract Brief exposure to low (0oC) or high (40oC) temperature elicits a protective response that prevents injury when the flesh fly, Sarcophaga crassipalpis Macquart, is subjected to more severe cold (‐10oC) or heat (45oC). Both the low and high temperature responses were found in all developmental stages of the fly, but were most pronounced in the pupal and pharate adult stages. The protective responses generated by brief exposure to 0 or 40oC appear similar in that both result in a rapid acquisition of cold or heat tolerance and a loss of protection after the flies are returned to 25oC. The protection generated by chilling is obvious within 10 min of exposure to 0oC while a 30 min exposure to 40oC is required to induce the high temperature protection. High temperature protects against cold shock injury within a narrow range (around 36oC) but we have no evidence that low temperature can protect against heat injury. We previously demonstrated that the rapid increase in cold tolerance correlates with concomitant increases in glycerol concentration, but in this study we found no significant elevation in glycerol in heat‐shocked flies. Thus the physiological and biochemical bases for the rapid responses to cold and heat appear to be different.

Defective gap-junctional communication associated with imaginal disc overgrowth and degeneration caused by mutations of the dco gene in Drosophila

The lethal(3)discs overgrown ( dco) locus of Drosophila melanogaster, located on the third chromosome at cytogenetic position 100A5,6–100B1,2, is necessary for normal development and growth control in the imaginal discs of the larva. Three recessive lethal alleles ( dco 2, dco 3 , and dco 18 ) in heteroallelic combinations and one allele ( dco 3 ) when homozygous cause the imaginal discs to continue to grow beyond the normal disc-intrinsic limit during an extended larval period. Some degeneration also occurs in the overgrowing discs. The discs overgrow even when transplanted early in their development into wild-type hosts, whereas normal discs stop growth at about the normal final size under such conditions, indicating that the overgrowth is a disc-autonomous effect of the mutations. During overgrowth the imaginal discs retain their single-layered epithelial structure except near regions of degeneration, and they differentiate into disc-appropriate but abnormal adult structures when transplanted into wild-type larval hosts. When the mutant larvae are reared under certain conditions a small percentage develop to the pharate adult stage, and these animals show a characteristic syndrome of abnormalities including swollen leg segments with many extra bristles, small or missing eyes, duplicated antennae and palpi, and separated vesicles of cuticle. A fourth recessive lethal allele ( dco le88 ), when homozygous or in heteroallelic combination with the overgrowth alleles, causes the imaginal discs to degenerate, producing a “discless” phenotype. Gap junction-mediated communication was assayed by observing the intercellular transfer of injected fluorescein complexon (dye coupling). Dye coupling in the imaginal discs of the dco genotypes that cause overgrowth was dramatically reduced at 4 days after egg laying (AEL) compared with wild-type controls. Coupling was more normal although still significantly reduced at 7–8 and 12–14 days AEL. In c43 hs1 , another disc overgrowth mutant, the imaginal disc cells also showed very reduced dye coupling at 4 days and incomplete coupling at 9 days. In contrast, discs from wild-type larvae, two other imaginal disc overgrowth mutants, and a cell death mutant showed extensive dye coupling at all stages tested. Electron microscopic morphometry revealed a reduction in gap-junction length per unit lateral plasma membrane length in dco 3 dco 18 and c43 hs1 wing discs, although not in dco 2 dco 3 , compared with wild-type wing discs. The results suggest that gap-junctional cell communication may be involved in the cell interactions that limit cell proliferation in vivo.

Ecdysteroids control vitellogenesis and egg maturation in pharate adult females of the Indianmeal moth, Plodia interpunctella

AbstractVitellogenesis occurs during the late pharate adult stage in the Indianmeal moth, Plodia interpunctella. Repeated treatment of pharate adult females with doses of 20‐hydroxyecdysone (20HE) from 10 to 250 ng per pupa suppressed oocyte growth and inhibited yolk protein accumulation in the oocytes. Treatment of the pharate adults with a biologically inactive ecdysteroid analogue, 22–isoecdysone, had no effect on egg maturation or yolk protein accumulation. The hormonal action of 20HE was not through the inhibition of the corpora allata or juvenile hormone levels, because treatment with a juvenile hormone analogue did not reverse the inhibition by 20HE treatment. Exposure of early vitellogenic ovaries to 20HE in organ cultures in vitro showed that 20HE had a direct effect on the ovarian synthesis of YP2. At 20HE concentrations below 10 nM, YP2 synthesis was minimal, at 10 nM 20HE YP2 synthesis was maximal, and at concentrations higher than 10 nM YP2 synthesis was suppressed to 35% of the maximal level. Synthesis of most other ovarian proteins remained constant with the changing 20HE concentrations. Ovarian RNA from treated females translated in a reticulocyte lysate demonstrated that the hormonal effect of 20HE on the ovarian tissues was on the specific accumulation of translatable YP2 transcript as well as transcripts for a few other polypeptides. This study shows that 20HE controls the rate of egg development during metamorphosis and that declining titers of 20HE regulate the expression of adult genes.

Development of spermiduct and seminal vesicle during pharate adult of the cabbage armyworm,Mamestra brassicae, (Lepidoptera; Noctuidae)

Summary Development of the upper vas deferens and seminal vesicle, and the ecdysteroid titers of the cabbage armyworm (Mamestra brassicae) during pharate adult stage were investigated to provide evidence of endogenous control of the sperm movement through the reproductive tract. Apparently, development of the upper vas deferens is initiated when ecdysteroid titers increase after pupation and continues until eclosion. Sperm movement correlates with the decrease of ecdysteroid titers. When ecdysteroid titers remained at low levels, sperm release from the testis was observed one day before eclosion.

Mutations at the fat locus interfere with cell proliferation control and epithelial morphogenesis in Drosophila

Lethal mutations at the fat locus in Drosophila cause imaginal discs to continue to grow by cell proliferation far beyond their normal final size. During a greatly extended larval period, the overgrowing imaginal discs develop additional folds and lobes, but retain a single-layered epithelial structure. In the wing disc, the additional lobes originate in the proximal fold area, and in the extra tissue the cells are less columnar than normal. Mutant disc cells lack zonulae adherents as well as associated microtubules and microfilaments, and they show an abnormal distribution and reduced density of gap junctions. The effect on growth is disc-autonomous as shown by transplantation experiments. The overgrown imaginal discs retain the ability to differentiate adult cuticular structures, as shown by metamorphosis of discs after transplantation into wild-type larval hosts and by the ability of some mutant animals to develop to the pharate adult stage. The structures differentiated by mutant discs show many abnormalities including ingrowths, outgrowths, separated cuticular vesicles, and areas of reversed bristle polarity; some of these abnormalities suggest that the mutations interfere with cell adhesion as well as the control of cell proliferation. The fat locus is located in cytogenetic interval 24D5.6–7, and 18 alleles are known including spontaneous, chemically induced, X-ray-induced, and dysgenic mutations.

The Drosophila abelson proto-oncogene homolog: Identification of mutant alleles that have pleiotropic effects late in development

The Abelson gene in Drosophila ( abl) consists of ten exons extending over 26 kb of genomic DNA. The DNA sequence encodes a protein of 1520 amino acids with sequence homology to the human c- abl proto-oncogene product, beginning at the amino terminus and extending 656 amino acids through the region essential for tyrosine kinase activity. Mutant lesions in the abl gene were identified first by their failure to complement chromosomal deletions that overlap the abl DNA sequence and then by rescue of the mutant phenotypes with an abl minigene in transgenic flies. Elimination of abl zygotic function by mutations produces some recessive lethality at the pharate adult pupal stage, and mutant adults with reduced longevity, reduced fecundity, and an irregular pattern of retinal cells.

Lipophorin in the larval and adult stages ofMusca domestica

The major Musca domestica hemolymph lipoprotein, lipophorin, was purified from larval and from adult animals. The housefly lipophorin is composed of two apoproteins, apolipophorin I (Mr ∽ 253,000) and apolipophorin II (Mr ∽ 85,000). The lipophorin contains about 3.9% carbohydrates and reacts positively with concanavalin A. The density of larval lipophorin is equal to 1.152 g/ml and of adult lipophorin to 1.106 g/ml. The amount of lipophorin per animal increases during the larval stage, is constant during pupal stage, and suffers a great reduction at the pharate adult stage. The amount of lipophorin remains stable during the whole first gonotrophic cycle of the housefly. Lipophorin is not detected in the eggs of this insect.

Effect of ethyl- p-chlorophenoxyisobutyrate (CPIB) on the fatty acid composition of Ceratitis capitata

1. The hypolipidemic agent ethyl- p-chlorophenoxyisobutyrate (CPIB) administered at a larval dietary level of 4% produces variations in the fatty acid composition of the triglyceride, free fatty acid, phospholipid and total lipid fractions in the dipteran Ceratitis capitata. 2. CPIB produces a decrease in the degree of unsaturation during the larval stage and an increase in this parameter at the adult stage and has no effect on the pharate adult stage. These variations suggest an action of the CPIB at the desaturase level. 3. Specific variations in particular fatty acids that can account for some physiological effects produced by CPIB were found. In particular, a decrease in the levels of palmitic, palmitoleic and linoleic acids could account for the diminishing fecundity elicited by CPIB in insects.

PHARATE-ADULT DIAPAUSE OF BARBARA COLFAXIANA (KFT.): DIFFERENTIATION OF 1- AND 2-YEAR DORMANCY

AbstractDiscovery of diapause in Barbara colfaxiana (Kft.) in the pharate-adult stage of development has allowed establishment of morphological characteristics to distinguish 1-year-diapause individuals from 2-year-diapause individuals at an earlier stage than previously possible. These morphological and some histological criteria are described. On the basis of morphological characteristics alone, 1- and 2-year-diapause individuals can be readily and reliably recognized by the unaided eye. The practical significance as well as the potential contribution of these findings to further research are pointed out.

Cytochrome P-450 in insects. 6. Age dependency and phenobarbital induction of cytochrome P-450, P-450 reductase, and monooxygenase activities in susceptible and resistant strains of Musca domestica

Development and phenobarbital (PB) induction of microsomal cytochrome P-450, cytochrome P-450 reductase, two epoxidation, and two O-demethylation activities were examined in chronologically timed populations of insecticide-susceptible (NAIDM) and -resistant (Rutgers) house flies. Measurements of these enzymes started with the pharate adult stage and ended 5 days following eclosion. Untreated insects of both strains had comparable reductase levels, whereas cytochrome P-450 and associated monooxygenase activities were 1.5-fold or more higher in Rutgers. Maximum induction, as well as toxicity, occurred at a lower PB concentration in NAIDM than Rutgers. The drug caused consistently higher increases in enzymes and activities within 12 hr of starting treatment in both strains. When PB was withdrawn from treated flies (both strains) 48 hr after treatment began, specific activities (product min −1 mg protein −1) in all enzymes returned to control values in 24 hr while metabolic capacity (product min −1 insect −1) achieved control values within 48 hr. The changes in turnover numbers (pmol product min −1 pmol P-450 −1), in conjunction with the differences in the monooxygenation of the four substrates, suggest that PB treatment induced both a quantitative and qualitative change in NAIDM monooxygenation but only a quantitative change in Rutgers monooxygenation.

Cytochrome P-450 in insects. 7. Age dependency and phenobarbital induction of cytochrome P-450, P-450 reductase, and monooxygenase activities in the black blow fly ( Phormia regina, Meigen)

Development and phenobarbital (PB) induction of microsomal cytochrome P-450, NADPH-cytochrome c ( P-450) reductase, two epoxidation, and two O-demethylation activities were examined in chronologically timed populations of female black blow flies ( Phormia regina, Meigen). Measurements of these enzymes started with the pharate adult stage and ended 5 days following eclosion. Induction occurred in all enzymes, even at 0.005% PB, and was maximum at 0.15%. Dramatic induction of the O-demethylation of 7-methoxy-4-methylcoumarin was observed in flies dosed with the maximum concentration of the drug. This monooxygenase activity increased to nearly 1400 times the level in control flies, whereas the other O-demethylation (methoxyresorufin) and the two epoxidation reactions exhibited considerably less change. Induction of the structural enzymes of this enzyme system were 10-fold for cytochrome P-450 and 5-fold for NADPH-cytochrome c ( P-450) reductase. These data suggest that PB induces several P-450's in the blow fly, particularly one bearing a high degree of specificity for 7-methoxy-4-methycoumarin.

Regulatory properties of adenylate cyclase preparations of pharate adult from the insect Ceratitis capitata

1. 1. Adenylate cyclase from the pharate adult stage of development of the insect Ceratitis capitata is a membrane-bound Me 2+-dependent enzyme. Mn 2+ was more effective in promoting the catalytic activity of the enzyme than Mg 2+. Calcium ions inhibited the enzyme activity in the presence of either Mg 2+ or Mn 2+. 2. 2. Activation of the enzyme by fluoride in the presence of Mg 2+ was higher than in the presence of Mn 2+. The presence of fluoride did not modify the affinity of the enzyme to the substrate but V max was increased. 3. 3. Regulatory properties of this enzyme from the insect pharate adult were different from those of adenylate cyclase from adult brain membranes. 4. 4. Thus, GTP increased slightly the Mg 2+-basal activity whereas it inhibited the Mn 2+-basal activity. 5. 5. GppNHp activated markedly the Mg 2+-basal activity; however, GppNHp in the presence of Mn 2+ exerted a dual effect, activation and inhibition, at low and high nucleotide concentrations, respectively. 6. 6. Octopamine did not affect the GTP-dependent enzyme activity, possibly due to the low presence of nervous system in the pharate adult stage of development.

Immature Stages of Leptopilina boulardi (Hymenoptera: Eucoilidae), a Protelean Parasite of Drosophila spp. (Diptera: Drosophilidae)

Leptopilina boulardi (Barbotin et al.), a larval parasitoid of Drosophila spp., possesses a stalked egg, eucoiliform first- and second-stage larvae, and hymenopteriform third-, fourth-, and fifth-stage larvae, followed by a pupal and pharate adult stage. Host age at oviposition significantly affects parasitoid development, whereas host development is in turn affected by the presence of the parasitoid larva and host age when parasitized. The fact that, regardless of sex, adults from hosts parasitized within a 2-h period emerge over 5 days suggests that a density-independent component influences development time.

Hormonal basis of adult eclosion in the gypsy moth (Lepidoptera: Lymantriidae)

Eclosion hormone was found to control the stereotypic adult eclosion behaviour of Lymantria dispar, the gypsy moth. A bioassay for hormonal activity was developed utilizing pharate adult females, and comparisons were made with the Manduca wing assay. The distribution of eclosion hormone activity was confined to the central nervous system tissues including the protocerebrum, corpora allata/corpora cardiaca complex, thoracic and the last abdominal ganglion. Haemolymph ecdysteroid titres were determined daily throughout pupal-adult development, and the peak activity period was found in 3–4 day pupae. Eclosion hormone activity in the brain and corpora allata/corpora cardiaca complex started to increase when the ecdysteroid titre dropped to background levels. Eclosion hormone in the brain peaked in the pharate adult stage, was released in the haemolymph 1 h prior to eclosion, which coincides with the depletion of activity in the retrocerebral complex, and fell to undetectable levels after the adult emerged.