Using the Drosoplzilu EcR-Bl cDNA as a probe, we have cloned the putative ecdysteroid receptor from the mealworm Tenehrio rnolitttr. 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 ligandbinding domains of the Drosophila receptor, respectively. Its expression pattern has been examined in the epidermis during the last larval instar and pupal stage of 7: rnolitor, 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.

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.

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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.

Five PCR fragments corresponding to a part of the DNA-binding domain of different hormone nuclear receptors were isolated from Tenebrio molitor mRNAs. The sequence identity of three of them with known Drosophila nuclear receptors strongly suggests that they are the Tenebrio orthologs of seven-up, DHR3 and b -FTZ-F1, and thus named Tmsvp, TmHR3 and TmFTZ-F1. The full-length sequences of the other two were established. TmHR78 is either a new receptor of the DHR78 family or the same gene which has evolved rapidly, particularly in the E domain. TmGRF belongs to the GCNF1 family and its in vitro translated product binds to the extended half site TCAAGGTCA with high affinity. The periods of expression of the corresponding transcripts in epidermal cells during Tenebrio metamorphosis were analyzed as a function of 20-hydroxyecdysone titers measured in the hemolymph of the animals taken for RNA extraction. Comparison of the expression profiles of these nuclear receptors with those observed during Drosophila metamorphosis revealed similar temporal correlations as a function of ecdysteroid variations, which further supported the sequence identity data for TmSVP, TmHR3, TmFTZ-F1 and TmHR78.Keywords: ecdysone; GCNF1; insect; metamorphosis; nuclear receptors.The pioneering work on chromosomal puffing induced by ecdysteroid in Drosophila salivary gland cells [1] gave rise to the idea that the molting hormone ecdysone triggered a regulatory cascade of gene expression. During the last decade, this hypothesis has been confirmed by molecular studies identifying the ecdysone receptor (EcR) [2] and several ecdysone-induced orphan members of the nuclear hormone receptor superfamily in Drosophila [3], and by developmental studies showing that many of these puff gene products are expressed hierarchically at various times after ecdysone action [4±6]. Such studies also suggest that in different tissues, the combinatorial expression cascade of these ecdysone-induced receptors may be different, thus contributing to the specificity of response to the hormone.The identification of several orthologs of Drosophila nuclear hormone receptors in other insect species supports the idea that the regulatory cascade found in the fruit fly could be conserved throughout this class. However, differences occur at the structural and transcriptional levels among different insect species studied so far [3], which could explain the differences observed in their life-cycles. Indeed, even in the apparently homogenous group of holometabolous insects, the metamorphic events present such striking differences in timing and cell fate among different orders that it is of interest to compare the cascade of molecular events triggered by ecdysone described for Drosophila with that of other metamorphosing insects. The identification of Drosophila EcR homologs in several other holometabolous species [7±15] supports this molecular diversity hypothesis. However, less is known concerning the homologs of other nuclear receptors characterized in Drosophila and thus it seems in...

Lma-P22 is a cuticular surface protein specific to the tergal gland secretion of Leucophaea maderae adult males which is ingested by females just before copulation. The complete Lma-P22 cDNA sequence was determined by RT-PCR using primers based on Edman degradation fragments. The recombinant protein expressed in Escherichia coli was recognized by an anti-Lma-P22 antibody. Northern blot analysis indicates that the corresponding mRNA is transcribed only in the epidermis of male tergites. Sequence analysis indicated that Lma-P22 deduced protein belongs to the lipocalin family. Lipocalins are extracellular proteins which carry hydrophobic compounds and some of them can bind sexual pheromone in vertebrates. Lma-P22 is the first example of a lipocalin-like protein involved in insect sexual behavior.z 1999 Federation of European Biochemical Societies.

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Pupal forewing epidermis of the Coleoptera, Tenebrio rnolitor, was used to develop an in vitro system to study the hormonal control of metamorphosis at the cellular and molecular levels. Exposure to 1 pM 20-hydroxyecdysone for 48 h caused the formation of a typical adult cuticle. Under these conditions the expression of ACP-20, an adult-specific cuticular gene, was fivefold higher than in absence of exogenous hormone. This stimulation was also observed when a higher level of 20-hydroxyecdysone was maintained, and prevented by protein inhibitors, indicating that 20-hydroxyecdysone does not act directly on this gene. Exposure to 20-hydroxyecdysone followed by exposure in hormone-free medium caused the cessation of this stimulation, showing the requirement of the 20-hydroxyecdysone continuous presence for stimulating ACP-20 gene expression. Thus, unlike the other cuticular protein genes so far studied, its expression is not repressed by 20-hydroxyecdysone, and does not need the decline in ecdysteroids titer.