White Puparium Research Articles

A Unified Protocol for CRISPR/Cas9-Mediated Gene Knockout in Tephritid Fruit Flies Led to the Recreation of White Eye and White Puparium Phenotypes in the Melon Fly.

Tephritid fruit flies are among the most invasive and destructive agricultural pests worldwide. Over recent years, many studies have implemented the CRISPR/Cas9 genome-editing technology to dissect gene functions in tephritids and create new strains to facilitate their genetics, management, and control. This growing literature allows us to compare diverse strategies for delivering CRISPR/Cas9 components into tephritid embryos, optimize procedures, and advance the technology to systems outside the most thoroughly studied species within the family. Here, we revisit five years of CRISPR research in Tephritidae and propose a unified protocol for candidate gene knockout in fruit flies using CRISPR/Cas9. We demonstrated the efficiency of our protocol by disrupting the eye pigmentation gene white eye (we) in the melon fly, Zeugodacus cucurbitae (Coquillett) (Diptera: Tephritidae). High rates of somatic and germline mutagenesis were induced by microinjecting pre-assembled Cas9-sgRNA complexes through the chorion of embryos at early embryogenesis, leading to the rapid development of new mutant lines. We achieved comparable results when targeting the we orthologue in the oriental fruit fly, Bactrocera dorsalis (Hendel) (Diptera: Tephritidae), illustrating the reliability of our methods when transferred to other related species. Finally, we functionally validated the recently discovered white pupae (wp) loci in the melon fly, successfully recreating the white puparium phenotype used in suppression programs of this and other major economically important tephritids. This is the first demonstration of CRISPR-based genome-editing in the genus Zeugodacus, and we anticipate that the procedures described here will contribute to advancing genome-editing in other non-model tephritid fruit flies.

Genome-Wide Analysis of MicroRNAs in Relation to Pupariation in Oriental Fruit Fly.

Insect metamorphosis is a complex process involving drastic morphological and physiological changes. microRNAs (miRNAs) are a class of endogenous small non-coding RNAs that play key roles in regulating various biological processes, including metamorphosis, by post-transcriptional repression of mRNAs. The oriental fruit fly, Bactrocera dorsalis, is one of the most destructive insect pests in many Asian countries and the Pacific Islands. The regulatory role of miRNAs in B. dorsalis metamorphosis is unclear. To better understand the molecular regulatory mechanisms of miRNAs in pupariation, Illumina sequencing of the wandering stage (WS), the late WS and the white puparium stage of B. dorsalis were performed. Two hundred forty-nine miRNAs, including 184 known miRNAs and 65 novel miRNAs, were obtained. Among these miRNAs, 19 miRNAs were differentially expressed in pupariation, and eight miRNAs showed relative high expression levels (>50 TPM), of which five differentially expressed miRNAs (DEMs) had target differentially expressed genes (DEGs) predicted by the expected miRNA–mRNA negative regulation pattern using the Illumina HiSeq data. Four sets of DEMs and their predicted target DEGs were confirmed by qPCR. Of the four miRNAs, two miRNAs were down-regulated: miR-981, which may target pdpc, and Bdo-novel-mir-55, which potentially regulates spsX1, psB/C, and chit3. The other two miRNAs were up-regulated: let-7a-3p, which possibly controls lap, and Bdo-novel-mir-24, which may regulate ipc and sp1/2. This study provides a useful resource to elucidate the regulatory role of miRNAs and understand the molecular mechanisms of metamorphosis.

RNA-seq analysis of gene expression changes during pupariation in Bactrocera dorsalis (Hendel) (Diptera: Tephritidae)

BackgroundThe oriental fruit fly, Bactrocera dorsalis (Hendel) has been considered to be one of the most important agricultural pest around the world. As a holometabolous insect, larvae must go through a metamorphosis process with dramatic morphological and structural changes to complete their development. To better understand the molecular mechanisms of these changes, RNA-seq of B. dorsalis from wandering stage (WS), late wandering stage (LWS) and white puparium stage (WPS) were performed.ResultsIn total, 11,721 transcripts were obtained, out of which 1914 genes (578 up-regulated and 1336 down-regulated) and 2047 genes (655 up-regulated and 1392 down-regulated) were found to be differentially expressed between WS and LWS, as well as between WS and WPS, respectively. Of these DEGs, 1862 and 1996 genes were successfully annotated in various databases. The analysis of RNA-seq data together with qRT-PCR validation indicated that during this transition, the genes in the oxidative phosphorylation pathway, and genes encoding P450s, serine protease inhibitor, and cuticular proteins were down-regulated, while the serine protease genes were up-regulated. Moreover, we found some 20-hydroxyecdysone (20E) biosynthesis and signaling pathway genes had a higher expression in the WS, while the genes responsible for juvenile hormone (JH) synthesis, degradation, signaling and transporter pathways were down-regulated, suggesting these genes might be involved in the process of larval pupariation in B. dorsalis. For the chitinolytic enzymes, the genes encoding chitinases (chitinase 2, chitinase 5, chitinase 8, and chitinase 10) and chitin deacetylase might play the crucial role in the degradation of insect chitin with their expressions significantly increased during the transition. Here, we also found that chitin synthase 1A might be involved in the chitin synthesis of cuticles during the metamorphosis in B. dorsalis.ConclusionsSignificant changes at transcriptional level were identified during the larval pupariation of B. dorsalis. Importantly, we also obtained a vast quantity of RNA-seq data and identified metamorphosis associated genes, which would all help us to better understand the molecular mechanism of metamorphosis process in B. dorsalis.

A comparative study of haemocytes in three cyclorrhaphous dipteran flies

The larval haemolymph of three cyclorrhaphous dipteran flies, Sarcophaga ruficornis Fab., Musca domestica L. and Chrysomya megacephala Fab., possesses five different types of haemocytes: prohaemocytes; plasmatocytes; granulocytes; spherulocytes; oenocytoids. In addition, vermicytes are found in the haemolymph of S. ruficornis and adipohaemocytes in the haemolymph of M. domestica. The total haemocyte counts in the three dipteran flies show an increasing trend throughout the larval stage, attaining a peak value in the freshly formed white puparium stage and declining thereafter. The differential haemocyte count shows a decline in the number of prohaemocytes with an increase in the number of plasmatocytes, granulocytes and spherulocytes.

Activities of natural methyl farnesoids on pupariation and metamorphosis of Drosophila melanogaster

Methyl farnesoate (MF) and juvenile hormone (JH III), which bind with high affinity to the receptors USP and MET, respectively, and bisepoxy JH III (bisJH III) were assessed for several activities during Drosophila larval development, and during prepupal development to eclosed adults. Dietary MF and JH III were similarly active, and more active than bisJH III, in lengthening larval development prior to pupariation. However, the order of activity was changed (JH III > bisJH III > MF) with respect to preventing prepupae from eclosing as normal adults, whether administered in the larval diet or as topically applied at the white puparium stage. If endogenous production of all three larval methyl farnesoids was suppressed by a strongly driven RNAi against HMGCR in the corpora allata cells, most larvae did not attain pupariation. Farnesol (which has no demonstrated life-necessary function in larval life except in corpora allata cells as a precursor to methyl farnesoid biosynthesis) when incorporated into the diet rescued attainment of pupariation in a dose-dependent manner, presumably by rescuing endogenous production of all three hormones. A more mild suppression of endogenous methyl farnesoid production enabled larval attainment of pupariation. However, in this background dietary MF had increased activity in preventing puparia from attaining normal adult eclosion. The physiological relevance of using exogenous methyl farnesoids to block prepupal development to normally eclosed adults was tested by, instead, protecting in prepupae the endogenous titer of methyl farnesoids. JH esterase normally increases during the mid-late prepupal stage, presumably to clear endogenous methyl farnesoids. When JH esterase was inhibited with an RNAi, it prevented attainment of adult eclosion. Cultured adult corpora allata from male and female Aedes aegypti released both MF and JH III, and the A. aegypti nuclear receptor USP bound MF with nanomolar affinity. These A. aegypti data support the use of Drosophila as a model for mosquitoes of the binding of secreted MF to USP.

Rosy Function Is Required for Juvenile Hormone Effects in Drosophila melanogaster

Application of a high dose of juvenile hormone (JH) III or its mimics (JHM) to Drosophila at the white puparium stage causes the formation of a pupal-like abdomen with few or no short bristles. We report here that the rosy (ry) gene encoding the enzyme xanthine dehydrogenase (XDH), which catalyzes the final two-step oxidation in purine catabolism, is required for this effect of JH on the epidermis. In ry506 (null allele) homozygotes or hemizygotes, JH III or pyriproxifen (a JHM) had little effect on abdominal bristle or cuticle formation, but disrupted the development of the central nervous system as in wild-type flies. Wild-type ry rescued the JH sensitivity of the abdominal epidermis in ry506 mutants. Inhibition of XDH activity phenocopied the ry null mutant's insensitivity to JH. Larvae fed on hypoxanthine or xanthine showed a decreased JH sensitivity. ry506 clones were sensitive to JH, indicating that ry is required non-cell autonomously for the JH effects. Normally JH applied at pupariation causes the aberrant reexpression of the transcription factor broad in the abdominal epidermis during adult development, but in the ry506 mutant most of the cells in the dorsal tergite showed no broad reexpression, indicating that ry is upstream of broad in the JH signaling pathway.

Mode of action of an insect neuropeptide leucopyrokinin (LPK) on pupariation in fleshfly ( Sarcophaga bullata) larvae (Diptera: Sarcophagidae)

An insect neuropeptide leucopyrokinin (LPK) (pQTSFTPRLamide) accelerates pupariation in wandering larvae of the fleshfly Sarcophaga bullata. The period of sensitivity to the action of LPK begins approximately 4 h before pupariation. Within this period the degree of acceleration of contraction into the shape of a puparium is practically independent of the age at which the larvae are injected, while acceleration of tanning is distinctly more age dependent. From ligation experiments we conclude that intact central innervation is essential for the action of LPK on puparial contraction, whereas central neurones take no part in mediation of LPK action on tanning of the cuticle. An analysis of tensiometric recordings of muscular activity revealed that the actual time of LPK accelerated puparial contraction coincides with the beginning of the immobilisation/retraction phase. LPK accelerates the switch from wandering behaviour to immobilisation/retraction behaviour but has no effect on the onset and duration of motor patterns that normally underlie puparial contraction in controls. The morphology of an accelerated puparium is normal but its formation is temporally dissociated from normal ‘contraction patterns’ that are performed a long time after the puparium has contracted. It means that neuromuscular activity of larvae accelerated by LPK does not cease upon formation of the white puparium, but continues until the whole motor programme of pupariation behaviour is completed. Apparently the peptide acts on the integument by stimulating it to contract and shrink, and no specific patterns of muscular contractions are needed to properly shape the puparium. This finding sheds a new light on our understanding of the mechanism of puparium formation.

The Drosophila melanogaster-related angiotensin-I-converting enzymes Acer and Ance--distinct enzymic characteristics and alternative expression during pupal development.

Drosophila melanogaster express two distinct angiotensin-I-converting enzymes (ACEs) called Ance and Acer, which display a high level of primary structure similarity. We have expressed Acer in the yeast Pichia pastoris and purified the recombinant enzyme with a view to developing biochemical tools to distinguish between Acer and Ance. Purified Acer and Ance expressed in yeast were used to raise anti-Acer Ig and anti-Ance Ig that specifically cross-reacted with the respective enzyme on immunoblotting, but did not act as specific inhibitors. Acer cleaves the C-terminal dipeptides from benzoylglycyl-histidyl-leucine and [Leu5]enkephalin, and Acer and Ance are both able to act as endopeptidases, releasing the C-terminal dipeptideamide from [Leu5]enkephalinamide. However, Acer hydrolyses this substrate at a slightly faster rate than [Leu5]enkephalin, whereas Ance hydrolyses the peptide with a free C-terminus with a kcat 15-fold higher than [Leu5]enkephalinamide. In addition, Acer did not cleave angiotensin I. In contrast, Ance hydrolysed 25% of this substrate at an 8-fold lower enzyme concentration. Furthermore, Acer did not hydrolyse the synthetic substrates Phe-Ser-Pro-Arg-Leu-Gly-Arg-Arg and Phe-Ser-Pro-Arg-Leu-Gly-Lys-Arg, two partially processed putative locustamyotropin precursors, under conditions where Ance produced 82% substrate hydrolysis. Acer was inhibited by captopril, trandolaprilat and enalaprilat, with apparent Ki values in the nanomolar range, whereas lisinopril and fosinoprilat were less potent. We show that the two Drosophila ACEs are alternatively expressed in stages P1 (white puparium)-P15 (eclosion) of pupal development; Ance is expressed predominantly during stages P4-P7, whereas the ACE activity expressed during stages P9-P12 is mainly due to Acer suggesting different roles for the two enzymes during pupal development.

Two different cDNAs code for l-DOPA decarboxylase in the white prepuparium and eclosion developmental stages of the insect Ceratitis capitata

In insects the DOPA decarboxylase gene ( Ddc) is expressed in the epidermis where dopamine derivatives promote cuticle sclerotization and the central nervous system participating in the synthesis of serotonin and dopamine. The temporal course of Ddc enzyme activity during the white prepuparium and eclosion developmental stages primarily reflects this role. Here we report that a cDNA clone for l-DOPA decarboxylase (EC 4.1.1.28) has been isolated from the white puparium stage of the insect Ceratitis capitata, the cDNA sequence has been determined and it corresponds to an mRNA of 1341 base pairs. The comparison of this cDNA sequence with that of the eclosion stage which has been previously isolated in our laboratory revealed that the cDNA from the eclosion stage codes for protein consisting of 431 amino acid (aa) residues whereas that from the white prepuparium stage for protein of 425 aa residues. In addition there is a region of 33 aa residues close to the N-terminal region of the deduced protein in which marked differences in the aa composition are observed. This result was further confirmed by Northern blot analysis in which mRNAs isolated from both developmental stages were only hybridized with their respective polymerase chain reaction synthesized stage specific probes. These results suggest that during development of the insect C. capitata two different mRNAs are produced at the white prepuparium and the eclosion stages, giving rise to two protein isoforms presumably in response to different signals.

Rearrangement of the tubulin and actin cytoskeleton during programmed cell death in Drosophila salivary glands

During larva-to-pupa metamorphosis Drosophila salivary glands undergo programmed cell death by autophagocytosis. Although ultrastructure of Drosophila salivary glands has been extensively studied in the past, little is known about mechanism of programmed cell death, especially the role of the cytoskeleton. In this paper we describe changes in microtubule and actin filament network compared to the progress of DNA fragmentation and redistribution of acid phosphatase. In feeding and wandering larvae microtubules and actin filaments form regular networks localized mostly along the plasma membrane. The first major rearrangement of microtubules and actin filaments occurred when larvae everted spiracles and the glands shifted their secretion from saliva to mucoprotein glue (stage L1). Microtubule cytoskeleton became denser and actin filaments concentrated along cell boundaries. At the same time nuclei flattened and migrated into the microtubule-rich layer near the basal membrane. In late prepupae (8-10 h after P1) the microtubule network became fainter, and actin filaments appeared frequently deeper in cytoplasm, gradually concentrating around nuclei. Simultaneously large patches of acid phosphatase activity surrounded nuclei and shortly thereafter chromosomal DNA began to fragment. During the final collapse of the gland (early pupae, 13.5 h after formation of white puparium) cellular fragments and autophagic vacuoles contained a continuous F-actin lining and the microtubule network displayed signs of extensive degradation. The results are consistent with the hypothesis that, in Drosophila salivary glands, extensive autophagic activities target nuclei for degradation; that this process occurs late in the course of programmed cell death; and that it directly involves cytoskeletal structures which are altered far earlier during the course of cell death.

L-Dopa decar☐ylase mRNA levels are correlated with the L-Dopa decar☐ylase protein profile during the development of the insect Ceratitis capitata

L-Dopa decar☐ylase (DDC) activity was determined throughout the life cycle of Ceratitis capitata. Basal levels of enzyme activity were observed in 4-day-old larvae with the activity peaking at white puparium and adult eclosion stages. The increasing enzyme activity observed at these stages, is matched by an increase of the L-dopa decar☐ylase protein, as shown by immunoblotting with a polyclonal antibody raised against DDC purified from the stage of eclosion of the insect Ceratitis capitata. Immunoprecipitation analysis revealed the presence of DDC in the in vitro polysomal and reticulocyte lysate translated products, obtained from white puparium and eclosion stages with no DDC detectable in 4-day-old larvae. The Northern blot analysis performed with a 4 kb HpaI fragment of Drosophila melanogaster Dopa decar☐ylase gene confirmed the results obtained by Western blot analysis and by immunoprecipitation of DDC in the in vitro translated products of 4-day-old larval white puparium and eclosion stages.

Translocation-Based Genetic Sexing System for the Oriental Fruit Fly (Diptera: Tephritidae) Based on Pupal Color Dimorphism

The autosomal recessive white puparium (wp) mutant was used to construct the first genetic sex sorting system in the oriental fruit fly, Bactrocera dorsalis (Hendel). The translocation stocks have females with the white puparium phenotype and puparia of males are the wild-type color, brown. Viability measurements of the 49-wp translocation strain indicate that fitness relative to the wild-type and white puparium strains was not lower than expected. Hatch rate and larval viability were lower for the translocation strain, but there were no significant differences between strains for eclosion rates. This translocation-based sex sorting system is the only automatic method currently available for separation of male and female oriental fruit flies in sterile insect release programs.

Ecdysteroid-mediated uptake of arylphorin by larval fat bodies of Calliphora vicina: Involvement and developmental regulation of arylphorin binding proteins

In the final larval instar of the blowfly Calliphora vicina the storage protein arylphorin is stage-specifically and selectively taken up by the fat body cells from the haemolymph. Previously, we have identified three arylphorin binding proteins (ABP 130, ABP 96, ABP 65) in the membrane fraction of late larvae and white puparia. Here we demonstrate that all ABPs derive from a single translation product (ABP 130) that we consider as arylphorin receptor precursor. The appearance of the ABPs is developmentally regulated throughout the last larval instar. While ABP 130 and ABP 96 appear at day 4, ABP 65 emerges at the end of last larval instar, coinciding with the rise in ecdysteroid titre and the beginning of arylphorin incorporation. We showed that 20HE induces stage-specifically arylphorin uptake and is responsible for the transformation of ABP 96 into ABP 65 plus a 30 kDa protein, which does not bind arylphorin.

Haemolymph ecdysteroid titres at metamorphosis in the fruit fly Ceratitis capitata: multiple peaks not apparent in whole body extracts

Abstract. The titre profile of haemolymph ecdysteroids in the fruit fly Ceratitis capitata was constructed from determinations of haemolymph taken from single animals and compared with ecdysteroid levels in whole body extracts. Larvae were sampled throughout the period from the late third instar larva until eclosion and were synchronized using a conspicuous behavioural marker (‘leaping’) that occurs at the end of the wandering period. Extracts of the whole body exhibit two peaks of ecdysteroids, one associated with pupariation (‘pupariation peak’ of other authors) and the other with pharate adult development. However, the ‘pupariation peak’ corresponded with three temporally distinct peaks in the titre of haemolymph ecdysteroids. The first peak occurs at the time of head retraction (lh before formation of the white puparium) and may represent the initial response to release of prothoracicotropic hormone. The second peak occurs 2–3 h later at the onset of tanning and may be responsible for induction of dopa‐decarboxylase. These peaks are brief (c. 2h each), whereas the third is much longer (16 h) and occupies most of the ‘pupariation peak’. The first two peaks have not been reported previously in any dipteran. Their existence illustrates that extremely brief pulses of ecdysteroids occur in vivo, apparently with profound developmental consequences. The fact that brief pulses in the haemolymph titre are not apparent in whole body extracts emphasizes that results obtained using the latter method must be interpreted with caution.

Linkage analysis of five new genetic markers of the oriental fruit fly, Bactrocera dorsalis (Diptera: Tephritidae).

Five new autosomal recessive genes are described in the oriental fruit fly, Bactrocera dorsalis (Hendel). These genetic markers are associated into three linkage groups. The matte (mt) gene is linked to the previously described mandarin red (ma) gene, and the white puparium (wp) gene is linked to the white eye (we) and amethyst (am) loci. The third designated linkage group has the yellow eye (ye) marker. The we/we homozygote is epistatic to ye/ye, and each is epistatic to am/am and ma/ma.

Identification of binding proteins involved in the stage-specific uptake of arylphorin by the fat body cells of Calliphora vicina

The larval serum protein arylphorin of Calliphora vicina is stage-specifically and selectively taken up from the haemolymph by the fat body cells of late larvae and white puparia. We give evidence for specific arylphorin binding sites in a membrane fraction referring to the characteristics of a “receptor”. Scatchard plot analysis revealed the existence of more than one binding site. By means of a combined ligand-immunoblotting method we identified three arylphorin binding proteins with apparent molecular weights of 65, 96, and 130 kDa. These polypeptides exhibit different patters of appearance throughout the last larval instar.

Studies on the enzymes involved in puparial cuticle sclerotization in Drosophila melanogaster.

The properties of cuticular enzymes involved in sclerotization of Drosophila melanogaster puparium were examined. The cuticle-bound phenoloxidase from the white puparium exhibited a pH optimum of 6.5 in phosphate buffer and oxidized a variety of catecholic substrates such as 4-methylcatechol, N-beta-alanyldopamine, dopa, dopamine, N-acetyldopamine, catechol, norepinephrine, 3,4-dihydroxyphenylglycol, 3,4-dihydroxybenzoic acid, and 3,4-dihydroxyphenylacetic acid. Phenoloxidase inhibitors such as potassium cyanide and sodium fluoride inhibited the enzyme activity drastically, but phenylthiourea showed marginal inhibition only. This result, coupled with the fact that syringaldazine served as the substrate for the insoluble enzyme, confirmed that cuticular phenoloxidase is of the "laccase" type. In addition, we also examined the mode of synthesis of the sclerotizing precursor, 1,2-dehydro-N-acetyldopamine. Our results indicate that this catecholamine derivative is biosynthesized from N-acetyldopamine through the intermediate formation of N-acetyldopamine quinone and N-acetyldopamine quinone methide as established for Sarcophaga bullata [Saul, S. and Sugumaran, M., F.E.B.S. Letters 251, 69-73 (1989)]. Accordingly, successful solubilization and fractionation of cuticular enzymes involved in the introduction of a double bond in the side chain of N-acetyldopamine indicated that they included o-diphenoloxidase, 4-alkyl-o-quinone:p-quinone methide isomerase, and N-acetyldopamine quinone methide:dehydro N-acetyldopamine isomerase and not any side chain desaturase.

Detection of haemocyte proteins in the integument of the developing Mediterranean fruit flyCeratitis capitata : Evidence that certain haemocyte proteins serve as tyrosine carriers.

Studies of the synthesis of integumental proteins during the feeding and non-feeding stages ofCeratitis capitata demonstrated stage specificity. The synthetic profile changed dramatically, showing a maximum of protein synthesis just before the larval wandering stage, followed by an abrupt decline. The comparison between synthetic and accumulation profiles indicated that some polypeptides must be internalized into the integument from the haemolymph. The major haemolymph proteins or arylphorins have already been documented to be incorporated into the integument. In the present work, we demonstrated the interalization of some haemocyte proteins into the integument. For that purpose, polyclonal antibodies were raised against total haemocyte proteins. Immunoblot analysis of haemocyte salt extractable proteins revealed that the protein bands at 36, 54, 58, 84, 110 and 130 kDa were immunoreactive with the total haemocyte antibodies. Cell-free protein synthesis, organ culture experiments and immunoblot analysis indicated that the 36-, 54- and 58-kDa polypeptides were synthesized only in the haemocytes and were probably internalized into the integument from the serum. The 36-kDa polypeptide was also demonstrated to be internalized into the fat body of white puparia. The immunofluorescence experiments suggested that the internalization of haemocyte proteins first occurs into the epidermal cells and then into the cuticle. The presence of haemocyte proteins in the integument was also demonstrated by immunofluorescence experiments in twoC. capitata mutants. These mutations affect the darkening and stiffening of the cuticle. The demonstration of 36-, 54- and 58-kDa haemocyte polypeptides in the integument reveals a hitherto unknown function of this cell type. Moreover, the demonstration of tyrosine binding to the 54- and 58-kDa polypeptides points to their potential involvement in the sclerotization process in the cuticle.

The cuticle proteins of Lucilia cuprina: Stage specificity and immunological relatedness

Proteins extracted from larval, pupal and adult cuticles of the sheep blowfly, Lucilia cuprina, have been compared electrophoretically and immunologically. Larval, pupal and adult cuticles each possess a largely unique set of cuticle proteins. Cuticles from the three larval instars contain some shared proteins; of the nine major 3rd instar larval cuticle proteins, three appear to be present in the 1st instar and four in the 2nd instar. Immunological evidence suggests that these proteins are specific to the integument (i.e. to the epidermis plus cuticle) and are not transported to the cuticle via the haemolymph. Pupariation involves a concerted crosslinking of the major larval cuticle proteins, such that most extractable protein is rendered insoluble within 24 h of white puparium formation. Protein extracted from pupal cuticle resolves into seven major bands and that from adult cuticle into nine major bands on native polyacrylamide gels. The electrophoretic profile of proteins extracted from the pupal cuticle (which remains unsclerotized) is relatively constant throughout pupal life. In contrast, the profiles of proteins extracted from the adult cuticle (which becomes sclerotized) change markedly during development. Regional variation in pupal and adult cuticle protein composition is also found. Immunological cross-reactivity is obtained between an antiserum raised against L. cuprina larval cuticle proteins and proteins from the larval cuticle of Drosophila melanogaster.

A Developmental Analysis of Ecdysteroids During the Metamorphosis ofDrosophila Melanogaster

Abstract A combination of ion suppression reverse phase high pressure liquid chromatography (RP-HPLC) and differential radioimmunoassay (RIA) was used to identify and quantify ecdysteroids from Drosophila melanogaster during the development of the white puparium to the adult. The whole body ecdysteroid titer revealed a large peak approximately 30 hours after pupariation. RP-HPLC-RIA analysis of the brain-ring gland complex secretion in vitro revealed the presence of: ecdysone, 20-deoxymakisterone A, and a yet uncharacterized low polarity ecdysteroid (LP-1). The metabolism of ecdysone and 20-deoxymakisterone A was followed by analysis of the ecdysteroids during development. The white puparium (0 hour) yielded a four-fold greater quantity of free 20-hydroxyecdysone and makisterone A than ecdysone and 20-deoxymakisterone A, respectively, indicating high 20-monooxygenase activity during this stage. Another metabolite of ecdysone, 20,26-dihydroxyecdysone, was present at a quantity intermediate between that of ...