The Drosophila ACP65A cuticle gene: deletion scanning analysis of cis-regulatory sequences and regulation by DHR38.

The regulatory sequences of the Drosophila ACP65A cuticle gene were analyzed in vivo in transgenic flies, using both fusion genes constructs and transposase-mediated deletions within a P element containing ACP65A regulatory sequences fused to the lacZ gene (deletion scanning). The sequences located between -594 and +161 are sufficient to confer both temporal and spatial expression specificities, indicating the presence of tissue-specific enhancers and response elements to hormone-induced factors. In addition, timing of expression and tissue-specificity appear to be controlled by distinct cis-regulatory elements, which suggests the existence of independent hormonal and tissue-specific signaling pathways. Gain and loss of function studies also implicate DHR38, the Drosophila homolog of the vertebrate NGFI-B-type nuclear receptors, as an important activator of the ACP65A gene.

Molecular characterization of Lma-p54, a new epicuticular surface protein in the cockroach Leucophaea maderae (Dictyoptera, oxyhaloinae).

The epicuticular surface protein Lma-p54 is imbedded in the "cuticular waxes" which cover the abdominal surface of the adult Leucophaea maderae. Natural Lma-p54 was purified and the complete cDNA sequence was determined by RT-PCR using primers based on Edman degradation fragments. Northern blot and in situ hybridization analyses showed that Lma-p54 was expressed in the adult abdominal epidermis and in the chemical sense organs of both sexes. Sequence alignment indicates that Lma-p54 is closely related to aspartic proteases (EC 3.4.23). However, there are critical amino acid substitutions at the level of the active site and, since no proteolytic activity was detected in the abdominal secretion, the protein is likely inactive. As an inactive aspartic protease, Lma-p54 is related to pregnancy-associated glycoproteins (PAGs) which still present a peptide-binding ability. According to recent experiments using whole tergal protein secretions, a role in intraspecific contact recognition was proposed for this surface protein.

Molecular characterization of a new adult male putative calycin specific to tergal aphrodisiac secretion in the cockroach Leucophaea maderae.

Lma-p18 is an epicuticular surface protein specific to the tergal gland aphrodisiac secretion of Leucophaea maderae adult males. Native Lma-p18 was purified and the complete cDNA sequence was determined by RT-PCR using primers based on Edman degradation fragments. Northern blot and in situ hybridization analyses showed that Lma-p18 is expressed exclusively in the anterior part of male tergal gland, which is exposed only during sexual behavior. Sequence analysis indicated that Lma-p18 belongs to the calycin superfamily and is very similar to Lma-p22, the first known male-specific tergal protein in L. maderae. Lma-p18 and Lma-p22 were proposed to bind different sexually attractive compounds as other calycins.

A novel chitin-binding protein from the vestimentiferan Riftia pachyptila interacts specifically with beta-chitin. Cloning, expression, and characterization.

A cDNA from Riftia pachyptila was cloned. It encodes a novel 21.3-kDa protein from the worm protective tube, named RCBP (for Riftia chitin-binding protein). On the basis of partial tube-peptide sequences previously obtained, experiments using reverse transcriptase-mediated polymerase chain reaction and rapid amplification of cDNA ends led to the complete cDNA sequence. Analysis of its deduced amino acid sequence shows the presence of two chitin-binding domains. These domains are closely related to type 2 chitin-binding domains that are restricted to the animal kingdom. We showed by affinity assay and immunogold labeling that RCBP is the first protein so far known that binds specifically beta-chitin and that is unable to bind the most common alpha-form found in chitin secreting animals. The RCBP mRNA was found to be present in specific epidermal cells from the worm body wall, but never in the chitin-secreting gland cells. This unexpected result clearly indicates that this tube protein is synthesized in specialized areas of the outer epithelium and that at least two different tissues are involved in this exoskeleton synthesis.

Characterization of a cDNA encoding RP43, a CUB-domain-containing protein from the tube of Riftia pachyptila (Vestimentifera), and distribution of its transcript.

A major 43 kDa protein from the protective tube of Riftia pachyptila (Vestimentifera), named RP43, was partly microsequenced after isolation by SDS/PAGE from the protein fraction of tubes collected around the hydrothermal vents at the East Pacific Rise. On the basis of the partial peptide sequences obtained, experiments using reverse-transcriptase-mediated PCR and rapid amplification of cDNA ends led to the complete cDNA sequence. Analysis of deduced amino acid sequence of RP43 showed the presence of CUB domains (100-110-residue-spanning domains first reported in the complement subcomponents C1r/C1s, epidermal-growth-factor-related sea urchin protein and bone morphogenetic protein 1) that seem to be involved in protein-protein and glycosaminoglycan-protein interactions. This peculiarity strongly suggests that RP43 might have a crucial role in tightening the different elements of the worm tube. However, the absence of chitin-binding motifs inclines us to favour a role in protein-protein interactions during assembly of the tube components. The RP43 mRNA was found to be present in specific epidermal cells from the worm body wall but never in the chitin-synthesizing gland cells. This unexpected result clearly indicates that the major tube protein is synthesized in specialized areas of the outer epithelium and that at least two different tissues are involved in the synthesis of the exoskeleton.

Molecular cloning and expression of Tenebrio molitor ultraspiracle during metamorphosis and in vivo induction of its phosphorylation by 20-hydroxyecdysone.

Using a RT-PCR approach, the Tenebrio molitor homologue of Drosophila Ultraspiracle (TmUSP) was characterized. Its DNA binding domain shows a degree of identity with those of the other insect USPs. However, the ligand binding domain is closer to those of retinoid X receptors. Using an antibody raised against DmUSP, Western blot analysis of proteins from epidermis and other tissues revealed five immunoreactive bands, corresponding to different phosphorylated forms of a unique polypeptide, as shown by lambda-phosphatase treatment. The nuclear form of TmUSP seems unphosphorylated. An in vivo 20-hydroxyecdysone treatment increases considerably and rapidly the phosphorylated forms of TmUSP. This post-translational modification may play a role in the 20-hydroxyecdysone response.

Development of the wing discs of Zophobas atratus under natural and experimental conditions: occurrence of a gradual larval-pupal commitment in the epidermis of tenebrionid beetles.

Using light and electron microscopy, we studied the development of the wing discs in the large beetle Zophobas atratus, under natural and experimental conditions. A reversible differentiation of the wing discs is usually observed during supernumerary instars of crowded larvae. Juvenile hormone analog (JHA) application during the wandering period or compelled experimental crowding during the larval-pupal switchover - or commitment - inhibits the onset of metamorphosis. Isolation, followed by recrowding, also induces the disc cells to secrete unusual cuticular material. Recrowding is able to trigger the reversal of metamorphosis during the 4-day period when larval-pupal commitment is taking place. Likewise, feeding behaviour which normally stops at commitment often recovers. Ecdysis of intermediate instar animals (prothetelic larvae) corroborates the occurrence of a temporal and spatial variation to commitment, unique to each organ. All these data lead us to consider this 4-day period, which we have called the C period or commitment period, extending from the wandering stage (the previous T period) to the crooked posture stage (i.e. from eyestage 4 to 7) as the physiological time during which the larval organs are gradually committed to differentiate into pupal organs.

Structure, organization and expression of two clustered cuticle protein genes during the metamorphosis of an insect, Tenebrio molitor.

A 4-kb DNA segment of Tenebrio molitor (Insecta, Coleoptera) genomic DNA containing two larval-pupal cuticular genes has been cloned and sequenced. These genes, transcribed in opposite directions, are related in DNA sequence and the proteins encoded are very similar. Each of them contains a single intron located inside the sequence encoding the signal peptide, and a conserved sequence at -200 bp from the mRNA start position. These similarities in sequence suggest that these genes have evolved by duplication followed by diversification and that they are members of a family of genes with a common ancestry. They are the first example of clustered genes in Tenebrio molitor.

Characterization of two new cuticular genes specifically expressed during the post-ecdysial molting period in Tenebrio molitor.

In a previous study, we have isolated a cDNA, TM-ACP17, coding for a post-ecdysial adult protein of Tenebrio molitor. After screening of a genomic library with TM-ACP17, we report isolation and sequencing of TM-ACP17 gene and a new gene, TM-LPCP29, coding for a larval-pupal protein. These two genes exhibit a common sequence of 15 nucleotides and a characteristic of most cuticular protein genes so far described: an intron interrupting the signal peptide. The deduced aa sequence of TM-LPCP29 exhibits a high percentage of Ala (26.5%) and Val (17.5%) and is highly hydrophobic. In the N-terminal part, the motif VAAPV is repeated ten times. Numerous histidine residues are present in the C- and N-terminal regions. A comparison is made with other cuticle protein sequences. Northern hybridization analysis showed that TM-LPCP29 is present during larval and mainly pupal post-ecdysial cuticle secretion. In-situ hybridization revealed that TM-LPCP29 mRNA is expressed in epidermis and not in muscles or fat body.

Cloning of two putative ecdysteroid receptor isoforms from Tenebrio molitor and their developmental expression in the epidermis during metamorphosis.

Using the Drosophila EcR-B1 cDNA as a probe, we have cloned the putative ecdysteroid receptor from the mealworm Tenebrio molitor. We have isolated two cDNAs with different 5' termini that contain a complete open reading frame. These two cDNAs encode two proteins with distinct N-terminal regions corresponding to two isoforms. The coleopteran receptor is obviously related to the ecdysteroid receptor of other insects, but shares only 89% and 61% amino acid identities with the DNA-binding and ligand-binding domains of the Drosophila receptor, respectively. Its expression pattern has been examined in the epidermis during the last larval instar and pupal stage of T. molitor, in correlation with the hemolymph ecdysteroid titer. Hybridizations revealed two transcripts of 7 kb and 6.5 kb detected in most stages during metamorphosis and corresponding to the A and B1 isoforms. These two mRNAs are highly evident just before the rise of each ecdysteroid peak both in prepupae and in pupae. They show almost the same expression pattern in epidermis except for the second part of the pupal stage, during which only the A isoform is detected.

Cloning and sequencing of a cDNA encoding a larval-pupal-specific cuticular protein in Tenebrio molitor (Insecta, Coleoptera). Developmental expression and effect of a juvenile hormone analogue.

A cDNA clone encoding a larval-pupal cuticular protein, named TMLPCP-22, has been isolated by screening a library in expression vector with a monoclonal antibody made against pupal cuticular proteins of Tenebrio molitor. Northern-blot and in situ hybridization analyses showed that the expression of TMLPCP-22 is regulated in a stage-specific and tissue-specific manner; the transcript was present during the secretion of preecdysial larval and pupal cuticles and was restricted to epidermal cells. No expression was observed during adult cuticle deposition. In supernumerary pupae obtained after application of a juvenile hormone analogue, which is known to inhibit the adult programme, TMLPCP-22 mRNA was expressed again, confirming its larval-pupal specificity.

Identification, sequence and mRNA expression pattern during metamorphosis of a cDNA encoding a glycine-rich cuticular protein in Tenebrio molitor.

The study of insect cuticular proteins and their sequences is of interest because they are involved in protein-protein and protein-chitin interactions which confer the mechanical properties and fine architecture of the cuticle. Moreover, in the coleopteran Tenebrio molitor there is a dramatic change in cuticular architecture between pre- and postecdysial secretion. We report the isolation, by differential screening, and the sequence characterization of a cDNA clone encoding a cuticular protein of T. molitor, ACP17. After insertion in the expression vector pEX1, the recognition of the fusion protein by an anti-cuticular monoclonal antibody confirmed the cuticular nature of ACP17. Northern hybridization analysis showed that ACP17 mRNA expression begins weakly 3 days before adult ecdysis and strongly increases during the secretion of postecdysial adult cuticle, with a maximum just after ecdysis. In situ hybridization revealed that the ACP17 mRNA is only present in the epidermis which secretes hard cuticle. The deduced amino acid (aa) composition exhibits a high content of Gly (28%) and Ala (20%) and, particularly, two poly(Gx) stretches separated by repetitive motifs with proline AAPVA. A comparison is made with other cuticle aa sequences.

The precocious commitment of wing Anlagen in Tenebrio molitor revealed by the addition of 20-hydroxyecdysone.

An injection of 20-hydroxyecdysone (10 mug per animal) 6-13 days after the moult of the last larval instar of Tenebrio molitor induces the development of prothetelic larvae and larval-pupal intermediates. The state of larval-pupal switchover, or commitment, is only disclosed at the time of injection of the moulting hormone. Prothetelic A and B larvae, with small and medium sized wing Anlagen, undergo another larval or pupal instar. Prothetelic C larvae with bigger Anlagen are unable to moult, but the adult programme is expressed. Ecdysed larval-pupal intermediates give more or less perfect adults, while unecdysed mealworms, imprisoned in their larval cuticle, also expressed the adult programme. The commitment of Tenebrio is not a global switchover because a significant asynchronisation is noted between the development of organs considered. Animal crowding induces a delay in the appearance of wing Anlagen.

Nucleotide sequence of an adult-specific cuticular protein gene from the beetle Tenebrio molitor: effects of 20-hydroxyecdysone on mRNA accumulation.

The accumulation of transcripts from two adult-specific cuticular genes (ACP-20 and ACP-22) is shown to be modified after addition of exogenous 20-hydroxyecdysone. In the continuous presence of high levels of the hormone, the expression of ACP-20 gene is significantly weaker than that of untreated controls, while ACP-22 expression is 2.5-fold increased. During active synthesis of the ACP messages, a 0.5 microg 20-hydroxyecdysone injection causes a rapid 2-fold increase in ACP-22 mRNA and is not able to repress ACP-20 mRNA accumulation. We conclude that these genes whose transcripts appear in an almost coordinated manner in epidermal cells during the moulting cycle are regulated by ecdysteroids in a different way. In order to undertake a functional dissection of the promoter regions of ACP-22 gene, we have isolated and sequenced a genomic clone. The sequence similarities with other cuticular protein genes are discussed.

cDNA cloning and deduced amino acid sequence of a major, glycine-rich cuticular protein from the coleopteran Tenebrio molitor. Temporal and spatial distribution of the transcript during metamorphosis.

In Coleoptera, the elytra (forewings), with a very hard and thick cuticle, protect the membranous and delicate hindwings against mechanical stress. We have isolated and characterized a cDNA encoding a major cuticle protein in Tenebrio molitor, named ACP-20. The deduced amino acid sequence is roughly tripartite, with two terminal glycine-rich domains and a central region showing pronounced similarities with some other hard cuticle proteins. Northern blot and in situ hybridization analyses reveal that ACP-20 gene expression is developmentally regulated since transcript accumulation occurs only in epidermal regions synthesizing hard cuticle and is restricted to the period of preecdysial adult cuticle deposition. Moreover, application of a juvenile hormone analogue prevents the appearance of the transcript, indicating that juvenile hormone, a key molecule involved in the control of insect metamorphosis, negatively regulates the expression of the ACP-20 gene.

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.

Characterization of a cDNA clone encoding a glycine-rich cuticular protein of Tenebrio molitor: developmental expression and effect of a juvenile hormone analogue.

The complete sequence of a cDNA clone, isolated from epidermal mRNA of Tenebrio molitor using a monoclonal antibody raised against an adult-specific cuticular antigen only present in the hard cuticle, was obtained after primer extension at the 5' end. From this cDNA sequence, the deduced protein encompasses 199 amino acids (including a signal peptide) with a total molecular weight of 20.7 kDa. The protein exhibits a bipartite structure: glycine-rich region located in its NH2-terminal part and a carboxy-terminal domain sharing homologies with other cuticular proteins of Orthoptera, Diptera and Lepidoptera. In-situ hybridization analysis shows that the corresponding mRNA is present only in epidermal cells secreting the adult fibrous cuticle destined to become heavily sclerotized. In supernumerary pupae obtained after the application of the juvenile hormone analogue (JHA) to newly ecdysed pupae, the mRNA was undetectable, indicating that JHA can prevent the switch to the adult programme. However, in pupal-adult intermediates, obtained when JHA is applied later, the mRNA is detected.

Morphogenesis of the wing Anlagen in the mealworm beetle tenebrio molitor during the last larval instar.

The wing Anlagen of Tenebrio develop from epidermal cells located on the lateral margins of meso- and metathoraces. Three to four days after larval ecdysis, these cells start to proliferate slowly, continuing to do so until day 13 which corresponds to the period of the pupal commitment of the remaining epidermis. The wing Anlagen cells then proliferate rapidly until day 18.5. Three days before pupal ecdysis, the mitotic index falls suddenly while 40% of the Anlagen cells disappear owing to cell degeneration. The sudden changes observed in the mitotic index are correlated with two hemolymphatic peaks in ecdysteroid levels. Anlagen of the forewings and hindwings show similar development except for cuticular secretion at the end of the instar which is greater on the upper face of the forewings. A comparison is made with imaginal discs and histoblasts described in other holometabolous insects.

Variability of ecdysteroid-induced cell cycle alterations in Drosophila Kc sublines.

The cell cycle of two lines isolated from Drosophila Kc cells was followed by flow cytofluorometry and cell counting. The first line is the 8-9K clone which grew in a medium supplemented with 5% serum; the second, named subline KcO, grew in a serum-free medium. The stationary phase is characterized by a G2 cell accumulation: 73% in the 8-9K clone and 50% in the KcO subline. When the medium was supplemented with the steroid moulting hormone 20-hydroxyecdysone, more than 90% of 8-9K cells and 65% of KcO cells were progressively arrested in G2. In the continuous presence of 20-hydroxyecdysone, most of the 8-9K cells remain G2-arrested; no massive G2 release into M was observed and only a few cells were able to divide. When treated for only 3 or 7 days, a transient release into M and proliferation occurred after hormone-free medium renewal, largely masked by G2 cell death. These results are discussed in comparison with other reports on cell cycle alteration induced by ecdysteroids.

The in vitro development of the pupal integument and the effects of ecdysteroids in Tenebrio molitor (Insecta, Coleoptera).

In order to study the pupal-adult metamorphosis of Tenebrio in vitro, pupal sternites of different ages were cultured in Landureau's medium and their development systematically observed by electron microscopy. In hormone-free medium, explants taken from young pupae do not secrete pupal postecdysial cuticle in vitro, and the epidermis spontaneously detaches from the pupal cuticle. On the contrary, explants taken from pharate adults continue to secrete adult preecdysial cuticle in vitro, and the epidermis never detaches from the cuticle. Ecdysterone in physiological concentrations (0.2 to 4 micrograms/ml) induces the secretion of a new cuticle in explants from young pupae but the epidermis remains undifferentiated. Ecdysone is necessary for the induction of some adult differentiation. Moreover, the quality of the cuticle secreted in vitro is increased by the addition of 2% foetal calf serum; the best results have thus far been obtained in a medium containing 0.2 microgram/ml ecdysone, 1 microgram/ml ecdysterone, and 2% foetal calf serum.